Methods and compositions for treating amyloid-related diseases

ABSTRACT

Methods, compounds, pharmaceutical compositions and kits are described for treating or preventing amyloid-related disease.

RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 11/316,694, filed Dec. 21, 2005, which, in turn,claims priority to U.S. Provisional Patent Application 60/638,819, filedon Dec. 22, 2004. U.S. patent application Ser. No. 11/316,694, filedDec. 21, 2005 is also a continuation-in-part of U.S. patent applicationSer. No. 10/871,514, filed Jun. 18, 2004, now U.S. Pat. No. 7,414,076,issued Aug. 19, 2008, which claims priority to U.S. Provisional PatentApplication No. 60/512,047, filed Oct. 17, 2003, and U.S. ProvisionalPatent Application No. 60/480,906, filed Jun. 23, 2003, all entitledMethods and Compositions for Treating Amyloid-Related Diseases. Thisapplication is also related to U.S. Provisional Application No.60/638,636, filed Dec. 22, 2004 and U.S. patent application Ser. No.10/871,365, filed Jun. 18, 2004, now U.S. Pat. No. 7,244,764, issuedJul. 17, 2007.

This application is also related to U.S. Provisional Patent ApplicationNo. 60/512,017, filed Oct. 17, 2003, U.S. Provisional Patent ApplicationNo. 60/480,918, filed Jun. 23, 2003, and U.S. patent application Ser.No. 10/871,613, filed Jun. 18, 2004, abandoned, all entitled Methods forTreating Protein Aggregation Disorders.

This application is related to U.S. Provisional Patent Application No.60/512,116, filed Oct. 17, 2003, U.S. Provisional Patent Application No.60/480,984, filed Jun. 23, 2003, and U.S. application Ser. No.10/871,549, filed Jun. 18, 2004, abandoned, all entitled PharmaceuticalFormulations of Amyloid-Inhibiting Compounds.

This application is related to U.S. Provisional Patent Application No.60/436,379, filed Dec. 24, 2002, U.S. Provisional Patent Application No.60/482,214, filed Jun. 23, 2003, entitled Combination Therapy for theTreatment of Alzheimer's Disease, U.S. patent application Ser. No.10/746,138, abandoned, filed Dec. 24, 2003, International PatentApplication No. PCT/CA2003/002011, filed Dec. 24, 2003, and U.S. patentapplication Ser. No. 10/871,537, filed Jun. 18, 2004, pending, entitledTherapeutic Formulations for the Treatment of Beta-Amyloid RelatedDiseases.

This application is related to U.S. Provisional Patent Application No.60/512,135, filed Oct. 17, 2003, U.S. Provisional Patent Application No.60/482,058, filed Jun. 23, 2003, both entitled Synthetic Process forPreparing Compounds for Treating Amyloidosis, and U.S. patentapplication Ser. No. 10/871,543, filed Jun. 18, 2004, now U.S. Pat. No.7,253,306, issued Aug. 7, 2007, entitled Improved Pharmaceutical DrugCandidates and Method for Preparation Thereof.

This application is related to U.S. Provisional Patent Application Ser.No. 60/512,018, filed on Oct. 17, 2003 and U.S. Provisional PatentApplication Ser. No. 60/480,928, filed on Jun. 23, 2003, and U.S. patentapplication Ser. No. 10/871,512, filed Jun. 18, 2004, pending, allentitled Methods and Compositions for Treating Amyloid- andEpileptogenesis-Associated Diseases.

This application is also related to Method for Treating Amyloidosis,U.S. patent application Ser. No. 08/463,548, now U.S. Pat. No.5,972,328.

The entire contents of each of these patent applications and patents arehereby expressly incorporated herein by reference including withoutlimitation the specification, claims, and abstract, as well as anyfigures, tables, or drawings thereof.

BACKGROUND

Amyloidosis refers to a pathological condition characterized by thepresence of amyloid fibrils. Amyloid is a generic term referring to agroup of diverse but specific protein deposits (intracellular orextracellular) which are seen in a number of different diseases. Thoughdiverse in their occurrence, all amyloid deposits have commonmorphologic properties, stain with specific dyes (e.g., Congo red), andhave a characteristic red-green birefringent appearance in polarizedlight after staining. They also share common ultrastructural featuresand common X-ray diffraction and infrared spectra. Amyloid-relateddiseases can either be restricted to one organ or spread to severalorgans. The first instance is referred to as “localized amyloidosis”while the second is referred to as “systemic amyloidosis.”

Some amyloid diseases can be idiopathic, but most of these diseasesappear as a complication of a previously existing disorder. For example,primary amyloidosis (AL amyloid) can appear without any other pathologyor can follow plasma cell dyscrasia or multiple myeloma.

Secondary amyloidosis is usually seen associated with chronic infection(such as tuberculosis) or chronic inflammation (such as rheumatoidarthritis). A familial form of secondary amyloidosis is also seen inother types of familial amyloidosis, e.g., Familial Mediterranean Fever(FMF). This familial type of amyloidosis is genetically inherited and isfound in specific population groups. In both primary and secondaryamyloidosis, deposits are found in several organs and are thusconsidered systemic amyloid diseases.

“Localized amyloidoses” are those that tend to involve a single organsystem. Different amyloids are also characterized by the type of proteinpresent in the deposit. For example, neurodegenerative diseases such asscrapie, bovine spongiform encephalitis, Creutzfeldt-Jakob disease, andthe like are characterized by the appearance and accumulation of aprotease-resistant form of a prion protein (referred to as AScr orPrP-27) in the central nervous system. Similarly, Alzheimer's disease,another neurodegenerative disorder, is characterized by neuritic plaquesand neurofibrillary tangles. In this case, the amyloid plaques found inthe parenchyma and the blood vessel is formed by the deposition offibrillar Aβ amyloid protein. Other diseases such as adult-onsetdiabetes (type II diabetes) are characterized by the localizedaccumulation of amyloid fibrils in the pancreas. Once these amyloidshave formed, there is no known, widely accepted therapy or treatmentwhich significantly dissolves amyloid deposits in situ, prevents furtheramyloid deposition or prevents the initiation of amyloid deposition.

Each amyloidogenic protein has the ability to undergo a conformationalchange and to organize into β-sheets and form insoluble fibrils whichmay be deposited extracellularly or intracellularly. Each amyloidogenicprotein, although different in amino acid sequence, has the sameproperty of forming fibrils and binding to other elements such asproteoglycan, amyloid P and complement component. Moreover, eachamyloidogenic protein has amino acid sequences which, althoughdifferent, show similarities such as regions with the ability to bind tothe glycosaminoglycan (GAG) portion of proteoglycan (referred to as theGAG binding site) as well as other regions which promote β-sheetformation. Proteoglycans are macromolecules of various sizes andstructures that are distributed almost everywhere in the body. They canbe found in the intracellular compartment, on the surface of cells, andas part of the extracellular matrix. The basic structure of allproteoglycans is comprised of a core protein and at least one, butfrequently more, polysaccharide chains (GAGs) attached to the coreprotein. Many different GAGs have been discovered including chondroitinsulfate, dermatan sulfate, keratan sulfate, heparin, and hyaluronan.

In specific cases, amyloid fibrils, once deposited, can become toxic tothe surrounding cells. For example, the Aβ fibrils organized as senileplaques have been shown to be associated with dead neuronal cells,dystrophic neurites, astrocytosis, and microgliosis in patients withAlzheimer's disease. When tested in vitro, oligomeric (soluble) as wellas fibrillar Aβ peptide was shown to be capable of triggering anactivation process of microglia (brain macrophages), which would explainthe presence of microgliosis and brain inflammation found in the brainof patients with Alzheimer's disease. Both oligomeric and fibrillar Aβpeptide can also induce neuronal cell death in vitro. See, e.g., M. P.Lambert, et al., Proc. Natl. Acad. Sci. USA 95, 6448-53 (1998).

In another type of amyloidosis seen in patients with type II diabetes,the amyloidogenic protein IAPP, when organized in oligomeric forms or infibrils, has been shown to induce β-islet cell toxicity in vitro. Hence,appearance of IAPP fibrils in the pancreas of type II diabetic patientscontributes to the loss of the β islet cells (Langerhans) and organdysfunction which can lead to insulinemia.

Another type of amyloidosis is related to β₂ microglobulin and is foundin long-term hemodialysis patients. Patients undergoing long termhemodialysis will develop β₂-microglobulin fibrils in the carpal tunneland in the collagen rich tissues in several joints. This causes severepains, joint stiffness and swelling.

Amyloidosis is also characteristic of Alzheimer's disease. Alzheimer'sdisease is a devastating disease of the brain that results inprogressive memory loss leading to dementia, physical disability, anddeath over a relatively long period of time. With the aging populationsin developed countries, the number of Alzheimer's patients is reachingepidemic proportions.

People suffering from Alzheimer's disease develop a progressive dementiain adulthood, accompanied by three main structural changes in the brain:diffuse loss of neurons in multiple parts of the brain; accumulation ofintracellular protein deposits termed neurofibrillary tangles; andaccumulation of extracellular protein deposits termed amyloid or senileplaques, surrounded by misshapen nerve terminals (dystrophic neurites)and activated microglia (microgliosis and astrocytosis). A mainconstituent of these amyloid plaques is the amyloid-β peptide (Aβ), a39-43 amino-acid protein that is produced through cleavage of theβ-amyloid precursor protein (APP). Extensive research has been conductedon the relevance of Aβ deposits in Alzheimer's disease, see, e.g.,Selkoe, Trends in Cell Biology 8, 447-453 (1998). Aβ naturally arisesfrom the metabolic processing of the amyloid precursor protein (“APP”)in the endoplasmic reticulum (“ER”), the Golgi apparatus, or theendosomal-lysosomal pathway, and most is normally secreted as a 40(“Aβ1-40”) or 42 (“Aβ1-42”) amino acid peptide (Selkoe, Annu. Rev. CellBiol. 10, 373-403 (1994)). A role for Aβ as a primary cause forAlzheimer's disease is supported by the presence of extracellular Aβdeposits in senile plaques of Alzheimer's disease, the increasedproduction of Aβ in cells harboring mutant Alzheimer's diseaseassociated genes, e.g., amyloid precursor protein, presenilin I andpresenilin II; and the toxicity of extracellular soluble (oligomeric) orfibrillar Aβ to cells in culture. See, e.g., Gervais, Eur. Biopharm.Review, 40-42 (Autumn 2001); May, DDT 6, 459-62 (2001). Althoughsymptomatic treatments exist for Alzheimer's disease, this diseasecannot be prevented or cured at this time.

Alzheimer's disease is characterized by diffuse and neuritic plaques,cerebral angiopathy, and neurofibrillary tangles. Plaque and bloodvessel amyloid is believed to be formed by the deposition of insolubleAβ amyloid protein, which may be described as diffuse or fibrillary.Both soluble oligomeric Aβ and fibrillar Aβ are also believed to beneurotoxic and inflammatory.

Another type of amyloidosis is cerebral amyloid angiopathy (CAA). CAA isthe specific deposition of amyloid-β fibrils in the walls ofleptomingeal and cortical arteries, arterioles and veins. It is commonlyassociated with Alzheimer's disease, Down's syndrome and normal aging,as well as with a variety of familial conditions related to stroke ordementia (see Frangione et al., Amyloid: J. Protein Folding Disord. 8,Suppl. 1, 36-42 (2001)).

Presently available therapies for treatment of β-amyloid diseases arealmost entirely symptomatic, providing only temporary or partialclinical benefit. Although some pharmaceutical agents have beendescribed that offer partial symptomatic relief, no comprehensivepharmacological therapy is currently available for the prevention ortreatment of, for example, Alzheimer's disease.

SUMMARY OF THE INVENTION

The present invention relates to the use of certain compounds in thetreatment of amyloid-related diseases. In particular, the inventionrelates to a method of treating or preventing an amyloid-related diseasein a subject comprising administering to the subject a therapeuticamount of a compound of the invention. The invention also pertains toeach of the novel compounds of the invention as described herein. Amongthe compounds for use in the invention are those according to thefollowing Formulae, such that, when administered, amyloid fibrilformation, organ specific dysfunction (e.g., neurodegeneration), orcellular toxicity is reduced or inhibited.

In one embodiment, the invention pertains, at least in part to compoundsof Formula I:

wherein:

R¹ is a substituted or unsubstituted cycloalkyl, heterocyclic, aryl,arylcycloalkyl, bicyclic or tricyclic ring, a bicyclic or tricyclicfused ring group, or a substituted or unsubstituted C₂-C₁₀ alkyl group;

R² is selected from a group consisting of hydrogen, alkyl,mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl,triazolyl, imidazolyl, benzothiazolyl, and benzoimidazolyl;

Y is SO₃ ⁻X⁺, OSO₃ ⁻X⁺, or SSO₃ ⁻X⁺;

X⁺ is hydrogen, a cationic group, or an ester-forming group (i.e., as ina prodrug, which are described elsewhere herein); and

each of L¹ and L² is independently a substituted or unsubstituted C₁-C₅alkyl group or absent, or a pharmaceutically acceptable salt thereof,provided that when R¹ is alkyl, L¹ is absent.

In another embodiment, the invention pertains, at least in part tocompounds of Formula II:

wherein:

R¹ is a substituted or unsubstituted cyclic, bicyclic, tricyclic, orbenzoheterocyclic group or a substituted or unsubstituted C₂-C₁₀ alkylgroup;

R² is hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl,aryl, arylalkyl, thiazolyl, triazolyl, imidazolyl, benzothiazolyl,benzoimidazolyl, or linked to R¹ to form a heterocycle;

Y is SO₃ ⁻X⁺, OSO₃ ⁻X⁺, or SSO₃ ⁻X⁺;

X⁺ is hydrogen, a cationic group, or an ester forming moiety;

m is 0 or 1;

n is 1, 2, 3, or 4;

L is substituted or unsubstituted C₁-C₃ alkyl group or absent, or apharmaceutically acceptable salt thereof, provided that when R¹ isalkyl, L is absent.

In yet another embodiment, the invention pertains, at least in part tocompounds of Formula III:

wherein:

A is nitrogen or oxygen;

R¹¹ is hydrogen, salt-forming cation, ester forming group, —(CH₂)_(x)-Q,or when A is nitrogen, A and R¹¹ taken together may be the residue of anatural or unnatural amino acid or a salt or ester thereof;

Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl, orbenzoimidazolyl;

x is 0, 1, 2, 3, or 4;

n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

R³, R^(3a), R⁴, R^(4a), R⁵, R^(5a), R⁶, R^(6a), R⁷ and R^(7a) are eachindependently hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl,cycloalkyl, aryl, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, cyano,halogen, amino, tetrazolyl, or two R groups on adjacent ring atoms takentogether with the ring atoms form a double bond, provided that one ofR³, R^(3a), R⁴, R^(4a), R⁵, R^(5a), R⁶, R^(6a), R⁷ and R^(7a) is amoiety of Formula IIIa:

wherein:

m is 0, 1, 2, 3, or 4;

R^(A), R^(B), R^(C), R^(D), and R^(E) are independently selected from agroup of hydrogen, halogen, hydroxyl, alkyl, alkoxyl, halogenated alkyl,mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, cyano, thiazolyl,triazolyl, imidazolyl, tetrazolyl, benzothiazolyl, and benzoimidazolyl;and pharmaceutically acceptable salts and esters thereof, provided thatsaid compound is not3-(4-phenyl-1,2,3,6-tetrahydro-1-pyridyl)-1-propanesulfonic acid.

In yet another embodiment, the invention pertains at least in part tocompounds of Formula IV:

wherein:

A is nitrogen or oxygen;

R¹¹ is hydrogen, salt-forming cation, ester forming group, —(CH₂)_(x)-Q,or when A is nitrogen, A and R¹¹ taken together may be the residue of anatural or unnatural amino acid or a salt or ester thereof;

Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl, orbenzoimidazolyl;

x is 0, 1, 2, 3, or 4;

n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

R⁴, R^(4a), R⁵, R^(5a), R⁶, R^(6a), R⁷, and R^(7a) are eachindependently hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl,cycloalkyl, aryl, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, cyano,halogen, amino, tetrazolyl, R⁴ and R⁵ taken together, with the ringatoms they are attached to, form a double bond, or R⁶ and R⁷ takentogether, with the ring atoms they are attached to, form a double bond;

m is 0, 1, 2, 3, or 4;

R⁸, R⁹, R¹⁰, R¹¹, and R¹² are independently selected from a group ofhydrogen, halogen, hydroxyl, alkyl, alkoxyl, halogenated alkyl,mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, cyano, thiazolyl,triazolyl, imidazolyl, tetrazolyl, benzothiazolyl, and benzoimidazolyl,and pharmaceutically acceptable salts and esters thereof.

In another embodiment, the invention includes compounds of Formula V:

wherein:

A is nitrogen or oxygen;

R¹¹ is hydrogen, salt-forming cation, ester forming group, —(CH₂)_(x)-Q,or when A is nitrogen, A and R¹¹ taken together may be the residue of anatural or unnatural amino acid or a salt or ester thereof;

Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl, orbenzoimidazolyl;

x is 0, 1, 2, 3, or 4;

n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

aa is a natural or unnatural amino acid residue;

m is 0, 1, 2, or 3;

R¹⁴ is hydrogen or protecting group;

R¹⁵ is hydrogen, alkyl or aryl, and pharmaceutically acceptable saltsand prodrugs thereof.

In another embodiment, the invention includes compounds of the FormulaVI:

wherein:

n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

A is oxygen or nitrogen;

R¹¹ is hydrogen, salt-forming cation, ester forming group, —(CH₂)_(x)-Q,or when A is nitrogen, A and R¹¹ taken together may be the residue of anatural or unnatural amino acid or a salt or ester thereof;

Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl, orbenzoimidazolyl;

x is 0, 1, 2, 3, or 4;

R¹⁹ is hydrogen, alkyl or aryl;

Y¹ is oxygen, sulfur, or nitrogen;

Y² is carbon, nitrogen, or oxygen;

R²⁰ is hydrogen, alkyl, amino, mercaptoalkyl, alkenyl, alkynyl,cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, tetrazolyl,imidazolyl, benzothiazolyl, or benzoimidazolyl;

R²¹ is hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl,aryl, arylalkyl, thiazolyl, triazolyl, tetrazolyl, imidazolyl,benzothiazolyl, benzoimidazolyl, or absent if Y² is oxygen;

R²² is hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl,aryl, arylalkyl, thiazolyl, triazolyl, tetrazolyl, imidazolyl,benzothiazolyl, benzoimidazolyl; or R²² is hydrogen, hydroxyl, alkoxy oraryloxy if Y¹ is nitrogen; or R²² is absent if Y¹ is oxygen or sulfur;or R²² and R²¹ may be linked to form a cyclic moiety if Y¹ is nitrogen;

R²³ is hydrogen, alkyl, amino, mercaptoalkyl, alkenyl, alkynyl,cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, tetrazolyl,imidazolyl, benzothiazolyl, or benzoimidazolyl, or absent if Y² isnitrogen or oxygen;

or pharmaceutically acceptable salts thereof.

In another embodiment, the invention includes compounds of Formula VII:

wherein:

n is 2, 3, or 4;

A is oxygen or nitrogen;

R¹¹ is hydrogen, salt-forming cation, ester forming group, —(CH₂)_(x)-Q,or when A is nitrogen, A and R¹¹ taken together may be the residue of anatural or unnatural amino acid or a salt or ester thereof;

Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl, orbenzoimidazolyl;

x is 0, 1, 2, 3, or 4;

G is a direct bond or oxygen, nitrogen, or sulfur;

z is 0, 1, 2, 3, 4, or 5;

m is 0 or 1;

R²⁴ is selected from a group consisting of hydrogen, alkyl,mercaptoalkyl, alkenyl, alkynyl, aroyl, alkylcarbonyl,aminoalkylcarbonyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl,imidazolyl, benzothiazolyl, and benzoimidazolyl;

each R²⁵ is independently selected from hydrogen, halogen, cyano,hydroxyl, alkoxy, thiol, amino, nitro, alkyl, aryl, carbocyclic, orheterocyclic, and pharmaceutically acceptable salts thereof.

In one embodiment, the compounds disclosed herein prevent or inhibitamyloid protein assembly into insoluble fibrils which, in vivo, aredeposited in various organs, or it favors clearance of pre-formeddeposits or slows deposition in patients already having deposits. Inanother embodiment, the compound may also prevent the amyloid protein,in its soluble, oligomeric form or in its fibrillar form, from bindingor adhering to a cell surface and causing cell damage or toxicity. Inyet another embodiment, the compound may block amyloid-induced cellulartoxicity or macrophage activation. In another embodiment, the compoundmay block amyloid-induced neurotoxicity or microglial activation. Inanother embodiment, the compound protects cells from amyloid inducedcytotoxicity of B-islet cells.

In another embodiment, the compound may enhance clearance from aspecific organ, e.g., the brain or it decreases concentration of theamyloid protein in such a way that amyloid fibril formation is preventedin the targeted organ.

The compounds of the invention may be administered therapeutically orprophylactically to treat diseases associated with amyloid fibrilformation, aggregation or deposition. The compounds of the invention mayact to ameliorate the course of an amyloid related disease using any ofthe following mechanisms (this list is meant to be illustrative and notlimiting): slowing the rate of amyloid fibril formation or deposition;lessening the degree of amyloid deposition; inhibiting, reducing, orpreventing amyloid fibril formation; inhibiting neurodegeneration orcellular toxicity induced by amyloid; inhibiting amyloid inducedinflammation; enhancing the clearance of amyloid; or favoring thedegradation of amyloid protein prior to its organization in fibrils.

The compounds of the invention may be administered therapeutically orprophylactically to treat diseases associated with amyloid-β fibrilformation, aggregation or deposition. The compounds of the invention mayact to ameliorate the course of an amyloid-β related disease using anyof the following mechanisms (this list is meant to be illustrative andnot limiting): slowing the rate of amyloid-β fibril formation ordeposition; lessening the degree of amyloid-β deposition; inhibiting,reducing, or preventing amyloid-β fibril formation; inhibitingneurodegeneration or cellular toxicity induced by amyloid-β; inhibitingamyloid-β induced inflammation; enhancing the clearance of amyloid-βfrom the brain; or favoring the degradation of amyloid-β protein priorto its organization in fibrils.

Therapeutic compounds of the invention may be effective in controllingamyloid-β deposition either following their entry into the brain(following penetration of the blood brain bather) or from the periphery.When acting from the periphery, a compound may alter the equilibrium ofAβ between the brain and the plasma so as to favor the exit of Aβ fromthe brain. It may also increase the catabolism of neuronal Aβ and changethe rate of exit from the brain. An increase in the exit of Aβ from thebrain would result in a decrease in Aβ brain and cerebral spinal fluid(CSF) concentration and therefore favor a decrease in Aβ deposition.Alternatively, compounds that penetrate the brain could controldeposition by acting directly on brain Aβ e.g., by maintaining it in anon-fibrillar form, favoring its clearance from the brain, or by slowingdown APP processing. These compounds could also prevent Aβ in the brainfrom interacting with the cell surface and therefore preventneurotoxicity, neurodegeneration or inflammation. They may also decreaseAβ production by activated microglia. The compounds may also increasedegradation by macrophages or neuronal cells.

In one embodiment, the method is used to treat Alzheimer's disease(e.g., sporadic, familial, or early AD). The method can also be usedprophylactically or therapeutically to treat other clinical occurrencesof amyloid-β deposition, such as in Down's syndrome individuals and inpatients with cerebral amyloid angiopathy (“CAA”) or hereditary cerebralhemorrhage.

In another embodiment, the method is used to treat mild cognitiveimpairment. Mild Cognitive Impairment (“MCI”) is a conditioncharacterized by a state of mild but measurable impairment in thinkingskills, which is not necessarily associated with the presence ofdementia. MCI frequently, but not necessarily, precedes Alzheimer'sdisease.

Additionally, abnormal accumulation of APP and of amyloid-β protein inmuscle fibers has been implicated in the pathology of sporadic inclusionbody myositis (IBM) (Askanas, et al., Proc. Natl. Acad. Sci. USA 93,1314-1319 (1996); Askanas, et al., Current Opinion in Rheumatology 7,486-496 (1995)). Accordingly, the compounds of the invention can be usedprophylactically or therapeutically in the treatment of disorders inwhich amyloid-beta protein is abnormally deposited at non-neurologicallocations, such as treatment of IBM by delivery of the compounds tomuscle fibers.

Additionally, it has been shown that Aβ is associated with abnormalextracellular deposits, known as drusen, that accumulate along the basalsurface of the retinal pigmented epithelium in individuals withage-related macular degeneration (AMD). AMD is a cause of irreversiblevision loss in older individuals. It is believed that Aβ depositioncould be an important component of the local inflammatory events thatcontribute to atrophy of the retinal pigmented epithelium, drusenbiogenesis, and the pathogenesis of AMD (Johnson, et al., Proc. Natl.Acad. Sci. USA 99(18), 11830-5 (2002)).

The present invention therefore relates to the use of compounds ofFormulae I, II, III, IV, V, VI, VII, or otherwise described herein inthe prevention or treatment of amyloid-related diseases, including,inter alia, Alzheimer's disease, cerebral amyloid angiopathy, mildcognitive impairment, inclusion body myositis, Down's syndrome, maculardegeneration, as well as other types of amyloidosis like IAPP-relatedamyloidosis (e.g., diabetes), primary (AL) amyloidosis, secondary (AA)amyloidosis and β₂ microglobulin-related (dialysis-related) amyloidosis.

In Type II diabetes related amyloidosis (IAPP), the amyloidogenicprotein IAPP induces β-islet cell toxicity when organized in oligomericforms or in fibrils. Hence, appearance of IAPP fibrils in the pancreasof type II diabetic patients contributes to the loss of the β isletcells (Langerhans) and organ dysfunction which leads to insulinemia.Primary amyloidosis (AL amyloid) is usually found associated with plasmacell dyscrasia and multiple myeloma. It can also be found as anidiopathic disease.

Secondary (AA) amyloidosis is usually seen associated with chronicinfection (such as tuberculosis) or chronic inflammation (such asrheumatoid arthritis). A familial form of secondary amyloidosis is alsoseen in Familial Mediterranean Fever (FMF). β₂ microglobulin-related(dialysis-related) amyloidosis is found in long-term hemodialysispatients. Patients undergoing long term hemodialysis will developβ₂-microglobulin fibrils in the carpal tunnel and in the collagen richtissues in several joints. This causes severe pains, joint stiffness andswelling. These deposits are due to the inability to maintain low levelsof β₂M in plasma of dialyzed patients. Increased plasma concentrationsof β₂M protein will induce structural changes and may lead to thedeposition of modified β₂M as insoluble fibrils in the joints.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of compounds of Formulae I, II,III, IV, V, VI, VII, or compounds otherwise described herein in thetreatment of amyloid-related diseases. For convenience, some definitionsof terms referred to herein are set forth below.

Amyloid-Related Diseases

AA (Reactive) Amyloidosis

Generally, AA amyloidosis is a manifestation of a number of diseasesthat provoke a sustained acute phase response. Such diseases includechronic inflammatory disorders, chronic local or systemic microbialinfections, and malignant neoplasms. The most common form of reactive orsecondary (AA) amyloidosis is seen as the result of long-standinginflammatory conditions. For example, patients with Rheumatoid Arthritisor Familial Mediterranean Fever (which is a genetic disease) can developAA amyloidosis. The terms “AA amyloidosis” and “secondary (AA)amyloidosis” are used interchangeably. AA fibrils are generally composedof 8,000 Dalton fragments (AA peptide or protein) formed by proteolyticcleavage of serum amyloid A protein (ApoSAA), a circulatingapolipoprotein which is mainly synthesized in hepatocytes in response tosuch cytokines as IL-1, IL-6 and TNF. Once secreted, ApoSAA is complexedwith HDL. Deposition of AA fibrils can be widespread in the body, with apreference for parenchymal organs. The kidneys are usually a depositionsite, and the liver and the spleen may also be affected. Deposition isalso seen in the heart, gastrointestinal tract, and the skin.

Underlying diseases which can lead to the development of AA amyloidosisinclude, but are not limited to inflammatory diseases, such asrheumatoid arthritis, juvenile chronic arthritis, ankylosingspondylitis, psoriasis, psoriatic arthropathy, Reiter's syndrome, AdultStill's disease, Behcet's syndrome, and Crohn's disease. AA deposits arealso produced as a result of chronic microbial infections, such asleprosy, tuberculosis, bronchiectasis, decubitus ulcers, chronicpyelonephritis, osteomyelitis, and Whipple's disease. Certain malignantneoplasms can also result in AA fibril amyloid deposits. These includesuch conditions as Hodgkin's lymphoma, renal carcinoma, carcinomas ofgut, lung and urogenital tract, basal cell carcinoma, and hairy cellleukemia. Other underlying conditions that may be associated with AAamyloidosis are Castleman's disease and Schnitzler's syndrome.

AL Amyloidoses (Primary Amyloidosis)

AL amyloid deposition is generally associated with almost any dyscrasiaof the B lymphocyte lineage, ranging from malignancy of plasma cells(multiple myeloma) to benign monoclonal gammopathy. At times, thepresence of amyloid deposits may be a primary indicator of theunderlying dyscrasia. AL amyloidosis is also described in detail inCurrent Drug Targets, 2004, 5 159-171.

Fibrils of AL amyloid deposits are composed of monoclonal immunoglobulinlight chains or fragments thereof. More specifically, the fragments arederived from the N-terminal region of the light chain (kappa or lambda)and contain all or part of the variable (V_(L)) domain thereof. Depositsgenerally occur in the mesenchymal tissues, causing peripheral andautonomic neuropathy, carpal tunnel syndrome, macroglossia, restrictivecardiomyopathy, arthropathy of large joints, immune dyscrasias,myelomas, as well as occult dyscrasias. However, it should be noted thatalmost any tissue, particularly visceral organs such as the kidney,liver, spleen and heart, may be involved.

Hereditary Systemic Amyloidoses

There are many forms of hereditary systemic amyloidoses. Although theyare relatively rare conditions, adult onset of symptoms and theirinheritance patterns (usually autosomal dominant) lead to persistence ofsuch disorders in the general population. Generally, the syndromes areattributable to point mutations in the precursor protein leading toproduction of variant amyloidogenic peptides or proteins. Table 1summarizes the fibril composition of exemplary forms of these disorders.

TABLE 1 Fibril Composition of Exemplary Amyloid-Related Diseases GeneticFibril Peptide/Protein Variant Clinical Syndrome ATTR protein fromTransthyretin Met30, many Familial amyloid polyneuropathy (FAP), andfragments others (Mainly peripheral nerves) ATTR protein fromTransthyretin Thr45, Ala60, Cardiac involvement predominant without andfragments Ser84, Met111, neuropathy, familial amyloid Ile122polyneuropathy, senile systemic amyloidosis, Tenosynovium N-terminalfragment of Arg26 Familial amyloid polyneuropathy (FAP), ApolipoproteinA1 (apoAI) (mainly peripheral nerves) N-terminal fragment of Arg26,Ostertag0type, non-neuropathic Apoliproprotein A1 (AapoAI) Arg50, Arg(predominantly visceral involvement) 60, others AapoAII fromApolipoprotein AII Familial amyloidosis Lysozyme (Alys) Thr56, His67Ostertag-type, non-neuropathic (predominantly visceral involvement)Fibrogen alpha chain fragment Leu554, Cranial neuropathy with latticcorneal Val526 dystrophy Gelsolin fragment (Agel) Asn187, Cranialneuropathy with lattice corneal Tyr187 dystrophy Cystatin C fragment(ACys) Glu68 Hereditary cerebral hemorrhage (cerebral amyloidangiopathy)-Icelandic type β-amyloid protein (Aβ) derived Gln693Hereditary cerebral hemorrhage (cerebral from Amyloid Precursor Protein(APP) amyloid angiopathy)-Dutch type β-amyloid protein (Aβ) derivedIle717, Phe717, Familial Alzheimer's Disease from Amyloid PrecursorProtein (APP) Gly717 β-amyloid protein (Aβ) derived Gln 618 Alzheimer'sdisease, Down's syndrome, from Amyloid Precursor Protein (APP),hereditary cerebral hemorrhage with e.g., bPP 695 amyloidosis, Dutchtype β-amyloid protein (Aβ) derived Asn670, Familial Dementia-probablyAlzheimer's from Amyloid Precursor Protein (APP) Leu671 Disease PrionProtein (PrP, APrP^(SC)) Leu102, Familial Creutzfeldt-Jakob disease;derived from Prp precursor Val167, Gerstmann-Sträussler-Scheinkersyndrome protein (51-91 insert) Asn178, (hereditary spongiformencephalopathies, Lys200 prion diseases) AA derived from Serum amyloidFamilial Mediterranean fever, predominant A protein (ApoSAA) renalinvolvement (autosomal recessive) AA derived from Serum amyloidMuckle-Well's syndrome, nephropathy, A protein (ApoSAA) deafness,urticaria, limb pain Unknown Cardiomyopathy with persistent atrialstandstill Unknown Cutaneous deposits (bullous, papular, pustulodermal)AH amyloid protein, derived Aγ I Myeloma associated amyloidosis fromimmunoglobulin heavy chain (gamma I) ACal amyloid protein from (Pro)Medullary carcinomas of the thyroid (pro)calcitonin calcitonin AANFamyloid protein from Isolated atrial amyloid atrial natriuretic factorApro from Prolactin Prolactinomas Abri/ADan from ABri peptide Britishand Danish familial Dementia Data derived from Tan S Y, Pepys M B.Amyloidosis. Histopathology, 25(5), 403-414 (November 1994), WHO/IUISNomenclature Subcommittee, Nomenclature of Amyloid and Amyloidosis.Bulletin of the World Health Organisation 1993; 71: 10508; and Merliniet al., Clin Chem Lab Med 2001; 39(11): 1065-75.

The data provided in Table 1 are exemplary and are not intended to limitthe scope of the invention. For example, more than 40 separate pointmutations in the transthyretin gene have been described, all of whichgive rise to clinically similar forms of familial amyloidpolyneuropathy.

In general, any hereditary amyloid disorder can also occur sporadically,and both hereditary and sporadic forms of a disease present with thesame characteristics with regard to amyloid. For example, the mostprevalent form of secondary AA amyloidosis occurs sporadically, e.g. asa result of ongoing inflammation, and is not associated with FamilialMediterranean Fever. Thus general discussion relating to hereditaryamyloid disorders below can also be applied to sporadic amyloidoses.

Transthyretin (TTR) is a 14 kiloDalton protein that is also sometimesreferred to as prealbumin. It is produced by the liver and choroidplexus, and it functions in transporting thyroid hormones and vitamin A.At least 50 variant forms of the protein, each characterized by a singleamino acid change, are responsible for various forms of familial amyloidpolyneuropathy. For example, substitution of proline for leucine atposition 55 results in a particularly progressive form of neuropathy;substitution of methionine for leucine at position 111 resulted in asevere cardiopathy in Danish patients.

Amyloid deposits isolated from heart tissue of patients with systemicamyloidosis have revealed that the deposits are composed of aheterogeneous mixture of TTR and fragments thereof, collectivelyreferred to as ATTR, the full length sequences of which have beencharacterized. ATTR fibril components can be extracted from such plaquesand their structure and sequence determined according to the methodsknown in the art (e.g., Gustaysson, A., et al., Laboratory Invest. 73:703-708, 1995; Kametani, F., et al., Biochem. Biophys. Res. Commun. 125:622-628, 1984; Pras, M., et al., PNAS 80: 539-42, 1983). Persons havingpoint mutations in the molecule apolipoprotein A1 (e.g., Gly→Arg26;Trp→Arg50; Leu→Arg60) exhibit a form of amyloidosis (“Östertag type”)characterized by deposits of the protein apolipoprotein AI or fragmentsthereof (AApoAI). These patients have low levels of high densitylipoprotein (HDL) and present with a peripheral neuropathy or renalfailure.

A mutation in the alpha chain of the enzyme lysozyme (e.g., Ile→Thr56 orAsp→His57) is the basis of another form of Östertag-type non-neuropathichereditary amyloid reported in English families. Here, fibrils of themutant lysozyme protein (Alys) are deposited, and patients generallyexhibit impaired renal function. This protein, unlike most of thefibril-forming proteins described herein, is usually present in whole(unfragmented) form (Benson, M. D., et al. CIBA Fdn. Symp. 199: 104-131,1996).

Immunoglobulin light chains tend to form aggregates in variousmorphologies, including fibrillar (e.g., AL amyloidosis and AHamyloidosis), granular (e.g., light chain deposition disease (LCDD),heavy chain deposition disease (HCDD), and light-heavy chain depositiondisease (LHCDD)), crystalline (e.g., Acquired Farconi's Syndrome), andmicrotubular (e.g., Cryoglobulinemia). AL and AH amyloidosis isindicated by the formation of insoluble fibrils of immunoglobulin lightchains and heavy chain, respectively, and/or their fragments. In ALfibrils, lambda (λ) chains such as λ VI chains (λ6 chains), are found ingreater concentrations than kappa (κ) chains. λIII chains are alsoslightly elevated. Merlini et al., Clin Chem Lab Med 39(11):1065-75(2001). Heavy chain amyloidosis (AH) is generally characterized byaggregates of gamma chain amyloid proteins of the IgG1 subclass. Eulitzet al., Proc Natl Acad Sci USA 87:6542-46 (1990).

Comparison of amyloidogenic to non-amyloidogenic light chains hasrevealed that the former can include replacements or substitutions thatappear to destabilize the folding of the protein and promoteaggregation. AL and LCDD have been distinguished from other amyloiddiseases due to their relatively small population monoclonal lightchains, which are manufactured by neoplastic expansion of anantibody-producing B cell. AL aggregates typically are well-orderedfibrils of lambda chains. LCDD aggregates are relatively amorphousaggregations of both kappa and lambda chains, with a majority beingkappa, in some cases κIV. Bellotti et al., Journal of Structural Biology13:280-89 (2000). Comparison of amyloidogenic and non-amyloidogenicheavy chains in patients having AH amyloidosis has revealed missingand/or altered components. Eulitz et al., Proc Natl Acad Sci USA87:6542-46 (1990) (pathogenic heavy chain characterized by significantlylower molecular mass than non-amyloidogenic heavy chains); and Solomonet al. Am J Hemat 45(2) 171-6 (1994) (amyloidogenic heavy chaincharacterized as consisting solely of the VH-D portion of thenon-amyloidogenic heavy chain).

Accordingly, potential methods of detecting and monitoring treatment ofsubjects having or at risk of having AL, LCDD, AH, and the like, includebut are not limited to immunoassaying plasma or urine for the presenceor depressed deposition of amyloidogenic light or heavy chains, e.g.,amyloid λ, amyloid κ, amyloid κIV, amyloid γ, or amyloid γ1.

Brain Amyloidosis

The most frequent type of amyloid in the brain is composed primarily ofAβ peptide fibrils, resulting in dementia associated with sporadic(non-hereditary) Alzheimer's disease. In fact, the incidence of sporadicAlzheimer's disease greatly exceeds forms shown to be hereditary.Nevertheless, fibril peptides forming plaques are very similar in bothtypes. Brain amyloidosis includes those diseases, conditions,pathologies, and other abnormalities of the structure or function of thebrain, including components thereof, in which the causative agent isamyloid. The area of the brain affected in an amyloid-related diseasemay be the stroma including the vasculature or the parenchyma includingfunctional or anatomical regions, or neurons themselves. A subject neednot have received a definitive diagnosis of a specifically recognizedamyloid-related disease. The term “amyloid related disease” includesbrain amyloidosis.

Amyloid-β peptide (“Aβ”) is a 39-43 amino acid peptide derived byproteolysis from a large protein known as Beta Amyloid Precursor Protein(“βAPP”). Mutations in βAPP result in familial forms of Alzheimer'sdisease, Down's syndrome, cerebral amyloid angiopathy, and seniledementia, characterized by cerebral deposition of plaques composed of Aβfibrils and other components, which are described in further detailbelow. Known mutations in APP associated with Alzheimer's disease occurproximate to the cleavage sites of β or γ-secretase, or within Aβ. Forexample, position 717 is proximate to the site of gamma-secretasecleavage of APP in its processing to Aβ, and positions 670/671 areproximate to the site of β-secretase cleavage. Mutations at any of theseresidues may result in Alzheimer's disease, presumably by causing anincrease in the amount of the 42/43 amino acid form of Aβ generated fromAPP. The familial form of Alzheimer's disease represents only 10% of thesubject population. Most occurrences of Alzheimer's disease are sporadiccases where APP and Aβ do not possess any mutation. The structure andsequence of Aβ peptides of various lengths are well known in the art.Such peptides can be made according to methods known in the art, orextracted from the brain according to known methods (e.g., Glenner andWong, Biochem. Biophys. Res. Comm. 129, 885-90 (1984); Glenner and Wong,Biochem. Biophys. Res. Comm. 122, 1131-35 (1984)). In addition, variousforms of the peptides are commercially available. APP is expressed andconstitutively catabolized in most cells. The dominant catabolic pathwayappears to be cleavage of APP within the Aβ sequence by an enzymeprovisionally termed α-secretase, leading to release of a solubleectodomain fragment known as APPsα. This cleavage precludes theformation of Aβ peptide. In contrast to this non-amyloidogenic pathway,APP can also be cleaved by enzymes known as β- and γ-secretase at the N-and C-termini of the Aβ, respectively, followed by release of Aβ intothe extracellular space. To date, BACE has been identified asβ-secretase (Vasser, et al., Science 286:735-741, 1999) and presenilinshave been implicated in γ-secretase activity (De Strooper, et al.,Nature 391, 387-90 (1998)). The 39-43 amino acid Aβ peptide is producedby sequential proteolytic cleavage of the amyloid precursor protein(APP) by the β and γ secretases enzyme. Although Aβ 40 is thepredominant form produced, 5-7% of total Aβ exists as Aβ42 (Cappai etal., Int. J. Biochem. Cell Biol. 31. 885-89 (1999)).

The length of the Aβ peptide appears to dramatically alter itsbiochemical/biophysical properties. Specifically, the additional twoamino acids at the C-terminus of Aβ 42 are very hydrophobic, presumablyincreasing the propensity of Aβ 42 to aggregate. For example, Jarrett,et al. demonstrated that Aβ42 aggregates very rapidly in vitro comparedto Aβ40, suggesting that the longer forms of Aβ may be the importantpathological proteins that are involved in the initial seeding of theneuritic plaques in Alzheimer's disease (Jarrett, et al., Biochemistry32, 4693-97 (1993); Jarrett, et al., Ann. N.Y. Acad. Sci. 695, 144-48(1993)).

This hypothesis has been further substantiated by the recent analysis ofthe contributions of specific forms of Aβ in cases of genetic familialforms of Alzheimer's disease (“FAD”). For example, the “London” mutantform of APP (APPV717I) linked to FAD selectively increases theproduction of Aβ 42/43 forms versus Aβ 40 (Suzuki, et al., Science 264,1336-40 (1994)) while the “Swedish” mutant form of APP (APPK670N/M671L)increases levels of both Aβ40 and Aβ42/43 (Citron, et al., Nature 360,672-674 (1992); Cai, et al., Science 259, 514-16, (1993)). Also, it hasbeen observed that FAD-linked mutations in the Presenilin-1 (“PS1”) orPresenilin-2 (“PS2”) genes will lead to a selective increase in Aβ42/43production but not Aβ40 (Borchelt, et al., Neuron 17, 1005-13 (1996)).This finding was corroborated in transgenic mouse models expressing PSmutants that demonstrate a selective increase in brain Aβ42 (Borchelt,op cit.; Duff, et al., Neurodegeneration 5(4), 293-98 (1996)). Thus theleading hypothesis regarding the etiology of Alzheimer's disease is thatan increase in Aβ42 brain concentration due to an increased productionand release of Aβ42 or a decrease in clearance (degradation or brainclearance) is a causative event in the disease pathology.

Multiple mutation sites in either Aβ or the APP gene have beenidentified and are clinically associated with either dementia orcerebral hemorrhage. Exemplary CAA disorders include, but are notlimited to, hereditary cerebral hemorrhage with amyloidosis of Icelandictype (HCHWA-I); the Dutch variant of HCHWA (HCHWA-D; a mutation in Aβ);the Flemish mutation of Aβ; the Arctic mutation of Aβ; the Italianmutation of Aβ; the Iowa mutation of Aβ; familial British dementia; andfamilial Danish dementia. CAA may also be sporadic.

As used herein, the terms “β amyloid,” “amyloid-β,” and the like referto amyloid β proteins or peptides, amyloid β precursor proteins orpeptides, intermediates, and modifications and fragments thereof, unlessotherwise specifically indicated. In particular, “Aβ” refers to anypeptide produced by proteolytic processing of the APP gene product,especially peptides which are associated with amyloid pathologies,including Aβ1-39, Aβ1-40, Aβ1-41, Aβ1-42, and Aβ1-43. For convenience ofnomenclature, “Aβ1-42” may be referred to herein as “Aβ(1-42)” or simplyas “Aβ42” or “Aβ₄₂” (and likewise for any other amyloid peptidesdiscussed herein). As used herein, the terms “β amyloid,” “amyloid-β,”and “Aβ” are synonymous.

Unless otherwise specified, the term “amyloid” refers to amyloidogenicproteins, peptides, or fragments thereof which can be soluble (e.g.,monomeric or oligomeric) or insoluble (e.g., having fibrillary structureor in amyloid plaque). See, e.g., M. P. Lambert, et al., Proc. Nat'lAcad. Sci. USA 95, 6448-53 (1998). “Amyloidosis” or “amyloid disease” or“amyloid-related disease” refers to a pathological conditioncharacterized by the presence of amyloid fibers. “Amyloid” is a genericterm referring to a group of diverse but specific protein deposits(intracellular or extracellular) which are seen in a number of differentdiseases. Though diverse in their occurrence, all amyloid deposits havecommon morphologic properties, stain with specific dyes (e.g., Congored), and have a characteristic red-green birefringent appearance inpolarized light after staining. They also share common ultrastructuralfeatures and common X-ray diffraction and infrared spectra.

Gelsolin is a calcium binding protein that binds to fragments and actinfilaments. Mutations at position 187 (e.g., Asp→Asn; Asp→Tyr) of theprotein result in a form of hereditary systemic amyloidosis, usuallyfound in patients from Finland, as well as persons of Dutch or Japaneseorigin. In afflicted individuals, fibrils formed from gelsolin fragments(Agel), usually consist of amino acids 173-243 (68 kDa carboxyterminalfragment) and are deposited in blood vessels and basement membranes,resulting in corneal dystrophy and cranial neuropathy which progressesto peripheral neuropathy, dystrophic skin changes and deposition inother organs. (Kangas, H., et al. Human Mol. Genet. 5(9): 1237-1243,1996). Other mutated proteins, such as mutant alpha chain of fibrinogen(AfibA) and mutant cystatin C (Acys) also form fibrils and producecharacteristic hereditary disorders. AfibA fibrils form depositscharacteristic of a nonneuropathic hereditary amyloid with renaldisease; Acys deposits are characteristic of a hereditary cerebralamyloid angiopathy reported in Iceland (Isselbacher, Harrison'sPrinciples of Internal Medicine, McGraw-Hill, San Francisco, 1995;Benson, et al.). In at least some cases, patients with cerebral amyloidangiopathy (CAA) have been shown to have amyloid fibrils containing anon-mutant form of cystatin C in conjunction with amyloid beta protein(Nagai, A., et al. Molec. Chem. Neuropathol. 33: 63-78, 1998).

Certain forms of prion disease are now considered to be heritable,accounting for up to 15% of cases, which were previously thought to bepredominantly infectious in nature. (Baldwin, et al., in ResearchAdvances in Alzheimer's Disease and Related Disorders, John Wiley andSons, New York, 1995). In hereditary and sporadic prion disorders,patients develop plaques composed of abnormal isoforms of the normalprion protein (PrP^(Sc)). A predominant mutant isoform, PrP^(Sc), alsoreferred to as AScr, differs from the normal cellular protein in itsresistance to protease degradation, insolubility after detergentextraction, deposition in secondary lysosomes, post-translationalsynthesis, and high β-pleated sheet content. Genetic linkage has beenestablished for at least five mutations resulting in Creutzfeldt-Jacobdisease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS), and fatalfamilial insomnia (FFI). (Baldwin, supra) Methods for extracting fibrilpeptides from scrapie fibrils, determining sequences and making suchpeptides are known in the art (e.g., Beekes, M., et al. J. Gen. Virol.76: 2567-76, 1995).

For example, one form of GSS has been linked to a PrP mutation at codon102, while telencephalic GSS segregates with a mutation at codon 117.Mutations at codons 198 and 217 result in a form of GSS in whichneuritic plaques characteristic of Alzheimer's disease contain PrPinstead of Aβ peptide. Certain forms of familial CJD have beenassociated with mutations at codons 200 and 210; mutations at codons 129and 178 have been found in both familial CJD and FFI. (Baldwin, supra).

Cerebral Amyloidosis

Local deposition of amyloid is common in the brain, particularly inelderly individuals. The most frequent type of amyloid in the brain iscomposed primarily of Aβ peptide fibrils, resulting in dementia orsporadic (non-hereditary) Alzheimer's disease. The most commonoccurrences of cerebral amyloidosis are sporadic and not familial Forexample, the incidence of sporadic Alzheimer's disease and sporadic CAAgreatly exceeds the incidence of familial AD and CAA. Moreover, sporadicand familial forms of the disease cannot be distinguished from eachother (they differ only in the presence or absence of an inheritedgenetic mutation); for example, the clinical symptoms and the amyloidplaques formed in both sporadic and familial AD are very similar, if notidentical.

Cerebral amyloid angiopathy (CAA) refers to the specific deposition ofamyloid fibrils in the walls of leptomingeal and cortical arteries,arterioles and veins. It is commonly associated with Alzheimer'sdisease, Down's syndrome and normal aging, as well as with a variety offamilial conditions related to stroke or dementia (see Frangione et al.,Amyloid: J. Protein Folding Disord. 8, Suppl. 1, 36-42 (2001)). CAA canoccur sporadically or be hereditary.

Senile Systemic Amyloidosis

Amyloid deposition, either systemic or focal, increases with age. Forexample, fibrils of wild type transthyretin (TTR) are commonly found inthe heart tissue of elderly individuals. These may be asymptomatic,clinically silent, or may result in heart failure. Asymptomaticfibrillar focal deposits may also occur in the brain (Aβ), corporaamylacea of the prostate (β₂ microglobulin), joints and seminalvesicles.

Dialysis-Related Amyloidosis (DRA)

Plaques composed of β₂ microglobulin (β₂M) fibrils commonly develop inpatients receiving long term hemodialysis or peritoneal dialysis. β₂microglobulin is a 11.8 kiloDalton polypeptide and is the light chain ofClass I MHC antigens, which are present on all nucleated cells. Undernormal circumstances, β₂M is usually distributed in the extracellularspace unless there is an impaired renal function, in which case β₂M istransported into tissues where it polymerizes to form amyloid fibrils.Failure of clearance such as in the case of impaired renal function,leads to deposition in the carpal tunnel and other sites (primarily incollagen-rich tissues of the joints). Unlike other fibril proteins, β₂Mmolecules are not produced by cleavage of a longer precursor protein andare generally present in unfragmented form in the fibrils. (Benson,supra). Retention and accumulation of this amyloid precursor has beenshown to be the main pathogenic process underlying DRA. DRA ischaracterized by peripheral joint osteoarthropathy (e.g., jointstiffness, pain, swelling, etc.). Isoforms of β₂M, glycated β₂M, orpolymers of β₂M in tissue are the most amyloidogenic form (as opposed tonative β₂M). Unlike other types of amyloidosis, β₂M is confined largelyto osteoarticular sites. Visceral depositions are rare. Occasionally,these deposits may involve blood vessels and other important anatomicsites.

Despite improved dialysis methods for removal of β₂M, the majority ofpatients have plasmatic β₂M concentrations that remain dramaticallyhigher than normal. These elevated β₂M concentrations generally lead toDiabetes-Related Amyloidosis (DRA) and comorbidities that contribute tomortality.

Islet Amyloid Polypeptide and Diabetes

Islet hyalinosis (amyloid deposition) was first described over a centuryago as the presence of fibrous protein aggregates in the pancreas ofpatients with severe hyperglycemia (Opie, E L., J. Exp. Med. 5: 397-428,1901). Today, islet amyloid, composed predominantly of islet amyloidpolypeptide (IAPP), or amylin, is a characteristic histopathologicalmarker in over 90% of all cases of Type II diabetes (also known asNon-Insulin Dependent Diabetes, or NIDDM). These fibrillar accumulationsresult from the aggregation of the islet amyloid polypeptide (IAPP) oramylin, which is a 37 amino acid peptide, derived from a largerprecursor peptide, called pro-IAPP.

IAPP is co-secreted with insulin in response to β-cell secretagogues.This pathological feature is not associated with insulin-dependent (TypeI) diabetes and is a unifying characteristic for the heterogeneousclinical phenotypes diagnosed as NIDDM (Type II diabetes).

Longitudinal studies in cats and immunocytochemical investigations inmonkeys have shown that a progressive increase in islet amyloid isassociated with a dramatic decrease in the population ofinsulin-secreting β-cells and increased severity of the disease. Morerecently, transgenic studies have strengthened the relationship betweenIAPP plaque formation and β-cell apoptosis and dysfunction, indicatingthat amyloid deposition is a principal factor in increasing severity ofType II diabetes.

IAPP has also been shown to induce β-islet cell toxicity in vitro,indicating that appearance of IAPP fibrils in the pancreas of Type II orType I diabetic patients (post-islet transplantation) could contributeto the loss of the β-cell islets (Langerhans) and organ dysfunction. Inpatients with Type II diabetes, the accumulation of pancreatic IAPPleads to formation of oligomeric IAPP, leading to a buildup ofIAPP-amyloid as insoluble fibrous deposits which eventually destroys theinsulin-producing β cells of the islet, resulting in β cell depletionand failure (Westermark, P., Grimelius, L., Acta Path. Microbiol.Scand., sect. A. 81: 291-300, 1973; de Koning, E J P., et al.,Diabetologia 36: 378-384, 1993; and Lorenzo, A., et al., Nature 368:756-760, 1994). Accumulation of IAPP as fibrous deposits can also havean impact on the ratio of pro-IAPP to IAPP normally found in plasma byincreasing this ratio due to the trapping of IAPP in deposits. Reductionof β cell mass can be manifested by hyperglycemia and insulinemia. Thisβ-cell mass loss can lead to a need for insulin therapy.

Diseases caused by the death or malfunctioning of a particular type ortypes of cells can be treated by transplanting into the patient healthycells of the relevant type of cell. This approach has been used for TypeI diabetes patients. Often pancreatic islet cells from a donor arecultured in vitro prior to transplantation, to allow them to recoverafter the isolation procedure or to reduce their immunogenicity.However, in many instances islet cell transplantation is unsuccessful,due to death of the transplanted cells. One reason for this poor successrate is IAPP, which organizes into toxic oligomers. Toxic effects mayresult from intracellular and extracellular accumulation of fibriloligomers. The IAPP oligomers can form fibrils and become toxic to thecells in vitro. In addition, IAPP fibrils are likely to continue to growafter the cells are transplanted and cause death or dysfunction of thecells. This may occur even when the cells are from a healthy donor andthe patient receiving the transplant does not have a disease that ischaracterized by the presence of fibrils. For example, compounds of thepresent invention may also be used in preparing tissues or cells fortransplantation according to the methods described in InternationalPatent Application (PCT) No. WO 01/003680.

The compounds of the invention may also stabilize the ratio of theconcentrations of Pro-IAPP/IAPP, pro-Insulin/Insulin and C-peptidelevels. In addition, as biological markers of efficacy, the results ofthe different tests, such as the arginine-insulin secretion test, theglucose tolerance test, insulin tolerance and sensitivity tests, couldall be used as markers of reduced β-cell mass and/or accumulation ofamyloid deposits. Such class of drugs could be used together with otherdrugs targeting insulin resistance, hepatic glucose production, andinsulin secretion. Such compounds might prevent insulin therapy bypreserving β-cell function and be applicable to preserving islettransplants.

Hormone-Derived Amyloidoses

Endocrine organs may harbor amyloid deposits, particularly in agedindividuals. Hormone-secreting tumors may also contain hormone-derivedamyloid plaques, the fibrils of which are made up of polypeptidehormones such as calcitonin (medullary carcinoma of the thyroid), andatrial natriuretic peptide (isolated atrial amyloidosis). Sequences andstructures of these proteins are well known in the art.

Miscellaneous Amyloidoses

There are a variety of other forms of amyloid disease that are normallymanifest as localized deposits of amyloid. In general, these diseasesare probably the result of the localized production or lack ofcatabolism of specific fibril precursors or a predisposition of aparticular tissue (such as the joint) for fibril deposition. Examples ofsuch idiopathic deposition include nodular AL amyloid, cutaneousamyloid, endocrine amyloid, and tumor-related amyloid. Other amyloidrelated diseases include those described in Table 1, such as familialamyloid polyneuropathy (FAP), senile systemic amyloidosis, Tenosynovium,familial amyloidosis, Ostertag-type, non-neuropathic amyloidosis,cranial neuropathy, hereditary cerebral hemorrhage, familial dementia,chronic dialysis, familial Creutzfeldt-Jakob disease;Gerstmann-Sträussler-Scheinker syndrome, hereditary spongiformencephalopathies, prion diseases, familial Mediterranean fever,Muckle-Well's syndrome, nephropathy, deafness, urticaria, limb pain,cardiomyopathy, cutaneous deposits, multiple myeloma, benign monoclonalgammopathy, maccoglobulinaemia, myeloma associated amyloidosis,medullary carcinomas of the thyroid, isolated atrial amyloid, anddiabetes.

The compounds of the invention may be administered therapeutically orprophylactically to treat diseases associated with amyloid fibrilformation, aggregation or deposition, regardless of the clinicalsetting. The compounds of the invention may act to ameliorate the courseof an amyloid related disease using any of the following mechanisms,such as, for example but not limited to: slowing the rate of amyloidfibril formation or deposition; lessening the degree of amyloiddeposition; inhibiting, reducing, or preventing amyloid fibrilformation; inhibiting amyloid induced inflammation; enhancing theclearance of amyloid from, for example, the brain; or protecting cellsfrom amyloid induced (oligomers or fibrillar) toxicity.

In an embodiment, the compounds of the invention may be administeredtherapeutically or prophylactically to treat diseases associated withamyloid-β fibril formation, aggregation or deposition. The compounds ofthe invention may act to ameliorate the course of an amyloid-β relateddisease using any of the following mechanisms (this list is meant to beillustrative and not limiting): slowing the rate of amyloid-β fibrilformation or deposition; lessening the degree of amyloid-β deposition;inhibiting, reducing, or preventing amyloid-β fibril formation;inhibiting neurodegeneration or cellular toxicity induced by amyloid-β;inhibiting amyloid-β induced inflammation; enhancing the clearance ofamyloid-β from the brain; or favoring greater catabolism of Aβ.

Compounds of the invention may be effective in controlling amyloid-βdeposition either following their entry into the brain (followingpenetration of the blood brain barrier) or from the periphery. Whenacting from the periphery, a compound may alter the equilibrium of Aβbetween the brain and the plasma so as to favor the exit of Aβ from thebrain. An increase in the exit of Aβ from the brain would result in adecrease in Aβ brain concentration and therefore favor a decrease in Aβdeposition. In addition, compounds that penetrate the brain may controldeposition by acting directly on brain Aβ, e.g., by maintaining it in anon-fibrillar form or favoring its clearance from the brain. Thecompounds may slow down APP processing; may increase degradation of Aβfibrils by macrophages or by neuronal cells; or may decrease Aβproduction by activated microglia. These compounds could also prevent Aβin the brain from interacting with the cell surface and thereforeprevent neurotoxicity, neurodegeneration, or inflammation.

In a preferred embodiment, the method is used to treat Alzheimer'sdisease (e.g., sporadic or familial AD). The method can also be usedprophylactically or therapeutically to treat other clinical occurrencesof amyloid-β deposition, such as in Down's syndrome individuals and inpatients with cerebral amyloid angiopathy (“CAA”), hereditary cerebralhemorrhage, or early Alzheimer's disease.

In another embodiment, the method is used to treat mild cognitiveimpairment. Mild Cognitive Impairment (“MCI”) is a conditioncharacterized by a state of mild but measurable impairment in thinkingskills, which is not necessarily associated with the presence ofdementia. MCI frequently, but not necessarily, precedes Alzheimer'sdisease. Additionally, abnormal accumulation of APP and of amyloid-βprotein in muscle fibers has been implicated in the pathology ofsporadic inclusion body myositis (IBM) (Askanas, V., et al. (1996) Proc.Natl. Acad. Sci. USA 93: 1314-1319; Askanas, V. et al. (1995) CurrentOpinion in Rheumatology 7: 486-496). Accordingly, the compounds of theinvention can be used prophylactically or therapeutically in thetreatment of disorders in which amyloid-beta protein is abnormallydeposited at non-neurological locations, such as treatment of IBM bydelivery of the compounds to muscle fibers.

Additionally, it has been shown that Aβ is associated with abnormalextracellular deposits, known as drusen, that accumulate along the basalsurface of the retinal pigmented epithelium in individuals withage-related macular degeneration (ARMD). ARMD is a cause of irreversiblevision loss in older individuals. It is believed that Aβ depositioncould be an important component of the local inflammatory events thatcontribute to atrophy of the retinal pigmented epithelium, drusenbiogenesis, and the pathogenesis of ARMD (Johnson, et al., Proc. Natl.Acad. Sci. USA 99(18), 11830-5 (2002)).

In another embodiment, the invention also relates to a method oftreating or preventing an amyloid-related disease in a subject(preferably a human) comprising administering to the subject atherapeutic amount of a compound according to the following Formulae orotherwise described herein, such that amyloid fibril formation ordeposition, neurodegeneration, or cellular toxicity is reduced orinhibited. In another embodiment, the invention relates to a method oftreating or preventing an amyloid-related disease in a subject(preferably a human) comprising administering to the subject atherapeutic amount of a compound according to the following Formulae orotherwise described herein, such that cognitive function is improved orstabilized or further deterioration in cognitive function is prevented,slowed, or stopped in patients with brain amyloidosis, e.g., Alzheimer'sdisease, Down's syndrome or cerebral amyloid angiopathy. These compoundscan also improve quality of daily living in these subjects.

The therapeutic compounds of the invention may treat amyloidosis relatedto type II diabetes by, for example, stabilizing glycemia, preventing orreducing the loss of β cell mass, reducing or preventing hyperglycemiadue to loss of β cell mass, and modulating (e.g., increasing orstabilizing) insulin production. The compounds of the invention may alsostabilize the ratio of the concentrations of pro-IAPP/IAPP.

The therapeutic compounds of the invention may treat AA (secondary)amyloidosis and/or AL (primary) amyloidosis, by stabilizing renalfunction, decreasing proteinuria, increasing creatinine clearance (e.g.,by at least 50% or greater or by at least 100% or greater), by leadingto remission of chronic diarrhea or weight gain (e.g., 10% or greater),or by reducing serum creatinine. Visceral amyloid content as determined,e.g., by SAP scintigraphy may also be reduced.

Compounds of the Invention

The present invention relates, at least in part, to the use of certainchemical compounds (and pharmaceutical formulations thereof) in theprevention or treatment of amyloid-related diseases, including, interalia, Alzheimer's disease, cerebral amyloid angiopathy, inclusion bodymyositis, Down's syndrome, diabetes related amyloidosis,hemodialysis-related amyloidosis (β₂M), primary amyloidosis (e.g., λ orκ chain-related), familial amyloid polyneuropathy (FAP), senile systemicamyloidosis, familial amyloidosis, Ostertag-type non-neuropathicamyloidosis, cranial neuropathy, hereditary cerebral hemorrhage,familial dementia, chronic dialysis, familial Creutzfeldt-Jakob disease,Gerstmann-Sträussler-Scheinker syndrome, hereditary spongiformencephalopathies, prion diseases, familial Mediterranean fever,Muckle-Well's syndrome, nephropathy, deafness, urticaria, limb pain,cardiomyopathy, cutaneous deposits, multiple myeloma, benign monoclonalgammopathy, maccoglobulinaemia, myeloma associated amyloidosis,medullary carcinomas of the thyroid, and isolated atrial amyloid.

The chemical structures herein are drawn according to the conventionalstandards known in the art. Thus, where an atom, such as a carbon atom,as drawn appears to have an unsatisfied valency, then that valency isassumed to be satisfied by a hydrogen atom even though that hydrogenatom is not necessarily explicitly drawn. The structures of some of thecompounds of this invention include stereogenic carbon atoms. It is tobe understood that isomers arising from such asymmetry (e.g., allenantiomers and diastereomers) are included within the scope of thisinvention unless indicated otherwise. That is, unless otherwisestipulated, any chiral carbon center may be of either (R)- or(S)-stereochemistry. Such isomers can be obtained in substantially pureform by classical separation techniques and bystereochemically-controlled synthesis. Furthermore, alkenes can includeeither the E- or Z-geometry, where appropriate. In addition, thecompounds of the present invention may exist in unsolvated as well assolvated forms with acceptable solvents such as water, THF, ethanol, andthe like. In general, the solvated forms are considered equivalent tothe unsolvated forms for the purposes of the present invention.

A “small molecule” refers to a compound that is not itself the productof gene transcription or translation (e.g., protein, RNA, or DNA) andpreferably has a low molecular weight, e.g., less than about 2500 amu.

In general, the term “nucleophile” is art-recognized to mean a chemicalgroup having a reactive pair of electrons that reacts with a compound bydisplacing a leaving group (commonly another nucleophile), such ascommonly occur in aliphatic chemistry as unimolecular (known as“S_(N)1”) or bimolecular (“S_(N)2”) reactions. Examples of nucleophilesinclude uncharged compounds such as amines, mercaptans, and alcohols,and charged groups such as alkoxides, thiolates, carbanions, and avariety of organic and inorganic anions. Illustrative anionicnucleophiles include, inter alia, simple anions such as azide, cyanide,thiocyanate, acetate, formate, or chloroformate, and bisulfite.Organometallic reagents such as organocuprates, organozincs,organolithiums, Grignard reagents, enolates, and acetylides, will underappropriate reaction conditions, be suitable nucleophiles.

Similarly, an “electrophile” means an atom, molecule, or ion able toaccept an electron pair, particularly a pair of electrons from anucleophile, such as typically occurs during an electrophilicsubstitution reaction. In an electrophilic substitution reaction, anelectrophile binds to a substrate with the expulsion of anotherelectrophile, e.g., the substitution of a proton by another electrophilesuch as a nitronium ion on an aromatic substrate (e.g., benzene).Electrophiles include cyclic compounds such as epoxides, aziridines,episulfides, cyclic sulfates, carbonates, lactones, and lactams; andnon-cyclic electrophiles include sulfates, sulfonates (e.g., tosylates),chlorides, bromides, and iodides. Generally, an electrophile may be asaturated carbon atom (e.g., a methylene group) bonded to a leavinggroup; however, an electrophile may also be an unsaturated group, suchas an aldehyde, ketone, ester, or conjugated (α,β-unsaturated) analogthereof, which upon reaction with a nucleophile forms an adduct.

The term “leaving group” generally refers to a group that is readilydisplaced and substituted by a nucleophile (e.g., an amine, a thiol, analcohol, or cyanide). Such leaving groups are well known and includecarboxylates, N-hydroxysuccinimide (“NHS”), N-hydroxybenzotriazole, ahalogen (fluorine, chlorine, bromine, or iodine), alkoxides, andthioalkoxides. A variety of sulfur-based leaving groups are routinelyused in synthetic chemistry, including alkane sulfonyloxy groups (e.g.,C₁-C₄ alkane such as methane sulfonyloxy, ethane sulfonyloxy, propanesulfonyloxy, and butane sulfonyloxy groups) and the halogenated analogs(e.g., halogeno(C₁-C₄ alkane) sulfonyloxy groups, such astrifluoromethane sulfonyloxy (i.e., triflate), 2,2,2-trichloroethanesulfonyloxy, 3,3,3-tribromopropane sulfonyloxy, and4,4,4-trifluorobutane sulfonyloxy groups), as well as arylsulfonyloxygroups (e.g., C₆-C₁₀ aryl optionally substituted with 1 to 3 C₁-C₄ alkylgroups, such as benzene sulfonyloxy, α-naphthylsulfonyloxy,β-naphthylsulfonyloxy, p-toluenesulfonyloxy (i.e., tosylates),4-tert-butylbenzene sulfonyloxy, mesitylene sulfonyloxy, and6-ethyl-α-naphthylsulfonyloxy groups).

“Activated esters” may be represented by the formula -COL, where L is aleaving group, typical examples of which includeN-hydroxysulfosuccinimidyl and N-hydroxysuccinimidyl groups; aryloxygroups substituted with electron-withdrawing groups (e.g., p-nitro,pentafluoro, pentachloro, p-cyano, or p-trifluoromethyl); and carboxylicacids activated by a carbodiimide to form an anhydride or mixedanhydride, e.g., —OCOR^(a) or —OCNR^(a)NHR^(b), where R^(a) and R^(b)are independently C₁-C₆ alkyl, C₅-C₈ alkyl (e.g., cyclohexyl), C₁-C₆perfluoroalkyl, or C₁-C₆ alkoxy groups. An activated ester may be formedin situ or may be an isolable reagent. Sulfosuccinimidyl esters,pentafluorothiophenol esters, and sulfotetrafluorophenol are preferredactivated esters. However, the ester leaving group may be, for example,substituted or unsubstituted C₁-C₆ alkyl (such as methyl, ethyl, propyl,isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, or hexyl), orsubstituted or unsubstituted C₆-C₁₄ aryl or heterocyclic groups, such as2-fluoroethyl, 2-chloroethyl, 2-bromoethyl, 2,2-dibromoethyl,2,2,2-trichloroethyl, 3-fluoropropyl, 4-chlorobutyl, methoxymethyl,1,1-dimethyl-1-methoxymethyl, ethoxymethyl, N-propoxymethyl,isopropoxymethyl, N-butoxymethyl, tert-butoxymethyl, 1-ethoxyethyl,1-methyl-1-methoxyethyl, 1-(isopropoxy)ethyl,3-methoxypropyl-4-methoxybutyl, fluoromethoxymethyl,2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl,3-fluoropropoxymethyl, 4-chlorobutoxyethyl, dibromomethoxyethyl,2-chloroethoxypropyl, fluoromethoxybutyl, 2-methoxyethoxymethyl,ethoxymethoxyethyl, methoxyethoxypropyl, methoxyethoxybutyl, benzyl,phenethyl, 3-phenylpropyl, 4-phenylbutyl, α-naphthylmethyl,β-naphthylmethyl, diphenylmethyl, triphenylmethyl,α-naphthyldipheylmethyl, 9-anthrylmethyl, 4-methylbenzyl,2,4,6-trimethylbenzyl, 3,4,5-trimethylbenzyl, 4-methoxybenzyl,4-methoxyphenyldiphenylmethyl, 2-nitrobenzyl, 4-nitrobenzyl,4-chlorobenzyl, 4-bromobenzyl, 4-cyanobenzyl,4-cyanobenzyldiphenylmethyl, or bis(2-nitrophenyl)methyl groups.

The term “electron-withdrawing group” is art-recognized and describesthe ability of a substituent to attract valence electrons (e.g.,pi-electrons) from neighboring atoms, e.g., the substituent is moreelectronegative than neighboring atoms, or it draws electrons to itselfmore than a hydrogen atom would at the same position. The Hammett sigmavalue (σ) is an accepted measure of a group's electron-donating andwithdrawing ability, especially the sigma para value (σ_(p)). See, e.g.,“Advanced Organic Chemistry” by J. March, 5^(th) Ed., John Wiley & Sons,Inc., New York, pp. 368-75 (2001). The Hammett constant values aregenerally negative for electron-donating groups (σ_(p)=−0.66 for NH₂)and positive for electron-withdrawing groups (σ_(p)=0.78 for a nitrogroup), σ_(p) indicating para substitution. Exemplaryelectron-withdrawing groups include nitro, acyl (ketone), formyl(aldehyde), sulfonyl, trifluoromethyl, halogeno (e.g., chloro andfluoro), and cyano groups, among others. Conversely, an“electron-donating group” designates a substituent that contributeselectrons more than hydrogen would if it occupied the same position inthe molecule. Examples include amino (including alkylamino anddialkylamino), aryl, alkoxy (including aralkoxy), aryloxy, mercapto andalkylthio, and hydroxyl groups, among others.

As used herein, “alkyl” groups include saturated hydrocarbons having oneor more carbon atoms, including straight-chain alkyl groups (e.g.,methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl,decyl, etc.), cyclic alkyl groups (or “cycloalkyl” or “alicyclic” or“carbocyclic” groups) (e.g., cyclopropyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl, etc.), branched-chain alkyl groups (isopropyl,tert-butyl, sec-butyl, isobutyl, etc.), and alkyl-substituted alkylgroups (e.g., alkyl-substituted cycloalkyl groups andcycloalkyl-substituted alkyl groups). The term “aliphatic group”includes organic moieties characterized by straight or branched-chains,typically having between 1 and 22 carbon atoms. In complex structures,the chains may be branched, bridged, or cross-linked. Aliphatic groupsinclude alkyl groups, alkenyl groups, and alkynyl groups.

In certain embodiments, a straight-chain or branched-chain alkyl groupmay have 30 or fewer carbon atoms in its backbone, e.g., C₁-C₃₀ forstraight-chain or C₃-C₃₀ for branched-chain. In certain embodiments, astraight-chain or branched-chain alkyl group may have 20 or fewer carbonatoms in its backbone, e.g., C₁-C₂₀ for straight-chain or C₃-C₂₀ forbranched-chain, and more preferably 18 or fewer. Likewise, preferredcycloalkyl groups have from 4-10 carbon atoms in their ring structure,and more preferably have 4-7 carbon atoms in the ring structure. Theterm “lower alkyl” refers to alkyl groups having from 1 to 6 carbons inthe chain, and to cycloalkyl groups having from 3 to 6 carbons in thering structure. Unless the number of carbons is otherwise specified,“lower” as in “lower aliphatic,” “lower alkyl,” “lower alkenyl,” etc. asused herein means that the moiety has at least one and less than about 8carbon atoms. In certain embodiments, a straight-chain or branched-chainlower alkyl group has 6 or fewer carbon atoms in its backbone (e.g.,C₁-C₆ for straight-chain, C₃-C₆ for branched-chain), and more preferably4 or fewer. Likewise, preferred cycloalkyl groups have from 3-8 carbonatoms in their ring structure, and more preferably have 5 or 6 carbonsin the ring structure. The term “C₁-C₆” as in “C₁-C₆ alkyl” means alkylgroups containing 1 to 6 carbon atoms.

Moreover, unless otherwise specified the term alkyl includes both“unsubstituted alkyls” and “substituted alkyls,” the latter of whichrefers to alkyl groups having substituents replacing one or morehydrogens on one or more carbons of the hydrocarbon backbone. Suchsubstituents may include, for example, alkenyl, alkynyl, halogeno,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano,amino (including alkyl amino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), imino, sulfhydryl, alkylthio,arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato,sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclic, alkylaryl, or aromatic (including heteroaromatic) groups.

An “arylalkyl” group is an alkyl group substituted with an aryl group(e.g., phenylmethyl (i.e., benzyl)). An “alkylaryl” moiety is an arylgroup substituted with an alkyl group (e.g., p-methylphenyl (i.e.,p-tolyl)). The term “n-alkyl” means a straight-chain (i.e., unbranched)unsubstituted alkyl group. An “alkylene” group is a divalent analog ofthe corresponding alkyl group. The terms “alkenyl” and “alkynyl” referto unsaturated aliphatic groups analogous to alkyls, but which containat least one double or triple carbon-carbon bond respectively. Suitablealkenyl and alkynyl groups include groups having 2 to about 12 carbonatoms, preferably from 2 to about 6 carbon atoms.

The term “aromatic group” or “aryl group” includes unsaturated andaromatic cyclic hydrocarbons as well as unsaturated and aromaticheterocycles containing one or more rings. Aryl groups may also be fusedor bridged with alicyclic or heterocyclic rings that are not aromatic soas to form a polycycle (e.g., tetralin). An “arylene” group is adivalent analog of an aryl group. Aryl groups can also be fused orbridged with alicyclic or heterocyclic rings which are not aromatic soas to form a polycycle (e.g., tetralin).

The term “heterocyclic group” includes closed ring structures analogousto carbocyclic groups in which one or more of the carbon atoms in thering is an element other than carbon, for example, nitrogen, sulfur, oroxygen. Heterocyclic groups may be saturated or unsaturated.Additionally, heterocyclic groups (such as pyrrolyl, pyridyl,isoquinolyl, quinolyl, purinyl, and furyl) may have aromatic character,in which case they may be referred to as “heteroaryl” or“heteroaromatic” groups.

Unless otherwise stipulated, aryl and heterocyclic (includingheteroaryl) groups may also be substituted at one or more constituentatoms. Examples of heteroaromatic and heteroalicyclic groups may have 1to 3 separate or fused rings with 3 to about 8 members per ring and oneor more N, O, or S heteroatoms. In general, the term “heteroatom”includes atoms of any element other than carbon or hydrogen, preferredexamples of which include nitrogen, oxygen, sulfur, and phosphorus.Heterocyclic groups may be saturated or unsaturated or aromatic.

Examples of heterocycles include, but are not limited to, acridinyl;azocinyl; benzimidazolyl; benzofuranyl; benzothiofuranyl;benzothiophenyl; benzoxazolyl; benzthiazolyl; benztriazolyl;benztetrazolyl; benzisoxazolyl; benzisothiazolyl; benzimidazolinyl;carbazolyl; 4aH-carbazolyl; carbolinyl; chromanyl; chromenyl;cinnolinyl; decahydroquinolinyl; 2H,6H-1,5,2-dithiazinyl;dihydrofuro[2,3-b]tetrahydrofuran; furanyl; furazanyl; imidazolidinyl;imidazolinyl; imidazolyl; 1H-indazolyl; indolenyl; indolinyl;indolizinyl; indolyl; 3H-indolyl; isobenzofuranyl; isochromanyl;isoindazolyl; isoindolinyl; isoindolyl; isoquinolinyl; isothiazolyl;isoxazolyl; methylenedioxyphenyl; morpholinyl; naphthyridinyl;octahydroisoquinolinyl; oxadiazolyl; 1,2,3-oxadiazolyl;1,2,4-oxadiazolyl; 1,2,5-oxadiazolyl; 1,3,4-oxadiazolyl; oxazolidinyl;oxazolyl; oxazolidinyl; pyrimidinyl; phenanthridinyl; phenanthrolinyl;phenazinyl; phenothiazinyl; phenoxathiinyl; phenoxazinyl; phthalazinyl;piperazinyl; piperidinyl; piperidonyl; 4-piperidonyl; piperonyl;pteridinyl; purinyl; pyranyl; pyrazinyl; pyrazolidinyl; pyrazolinyl;pyrazolyl; pyridazinyl; pyridooxazole; pyridoimidazole; pyridothiazole;pyridinyl; pyridyl; pyrimidinyl; pyrrolidinyl; pyrrolinyl; 2H-pyrrolyl;pyrrolyl; quinazolinyl; quinolinyl; 4H-quinolizinyl; quinoxalinyl;quinuclidinyl; tetrahydrofuranyl; tetrahydroisoquinolinyl;tetrahydroquinolinyl; tetrazolyl; 6H-1,2,5-thiadiazinyl;1,2,3-thiadiazolyl; 1,2,4-thiadiazolyl; 1,2,5-thiadiazolyl;1,3,4-thiadiazolyl; thianthrenyl; thiazolyl; thienyl; thienothiazolyl;thienooxazolyl; thienoimidazolyl; thiophenyl; triazinyl;1,2,3-triazolyl; 1,2,4-triazolyl; 1,2,5-triazolyl; 1,3,4-triazolyl; andxanthenyl. Preferred heterocycles include, but are not limited to,pyridinyl; furanyl; thienyl; pyrrolyl; pyrazolyl; pyrrolidinyl;imidazolyl; indolyl; benzimidazolyl; 1H-indazolyl; oxazolidinyl;benzotriazolyl; benzisoxazolyl; oxindolyl; benzoxazolinyl; and isatinoylgroups. Also included are fused ring and spiro compounds containing, forexample, the above heterocycles.

A common hydrocarbon aryl group is a phenyl group having one ring.Two-ring hydrocarbon aryl groups include naphthyl, indenyl,benzocyclooctenyl, benzocycloheptenyl, pentalenyl, and azulenyl groups,as well as the partially hydrogenated analogs thereof such as indanyland tetrahydronaphthyl. Exemplary three-ring hydrocarbon aryl groupsinclude acephthylenyl, fluorenyl, phenalenyl, phenanthrenyl, andanthracenyl groups.

Aryl groups also include heteromonocyclic aryl groups, i.e., single-ringheteroaryl groups, such as thienyl, furyl, pyranyl, pyrrolyl,imidazolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, andpyridazinyl groups; and oxidized analogs thereof such as pyridonyl,oxazolonyl, pyrazolonyl, isoxazolonyl, and thiazolonyl groups. Thecorresponding hydrogenated (i.e., non-aromatic) heteromonocyclic groupsinclude pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl,pyrazolidinyl, pyrazolinyl, piperidyl and piperidino, piperazinyl, andmorpholino and morpholinyl groups.

Aryl groups also include fused two-ring heteroaryls such as indolyl,isoindolyl, indolizinyl, indazolyl, quinolinyl, isoquinolinyl,phthalazinyl, quinoxalinyl, quinazolinyl, cinnolinyl, chromenyl,isochromenyl, benzothienyl, benzimidazolyl, benzothiazolyl, purinyl,quinolizinyl, isoquinolonyl, quinolonyl, naphthyridinyl, and pteridinylgroups, as well as the partially hydrogenated analogs such as chromanyl,isochromanyl, indolinyl, isoindolinyl, and tetrahydroindolyl groups.Aryl groups also include fused three-ring groups such as phenoxathiinyl,carbazolyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, phenoxazinyl, and dibenzofuranyl groups.

Some typical aryl groups include substituted or unsubstituted 5- and6-membered single-ring groups. In another aspect, each Ar group may beselected from the group consisting of substituted or unsubstitutedphenyl, pyrrolyl, furyl, thienyl, thiazolyl, isothiaozolyl, imidazolyl,triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isooxazolyl, pyridinyl,pyrazinyl, pyridazinyl, and pyrimidinyl groups. Further examples includesubstituted or unsubstituted phenyl, 1-naphthyl, 2-naphthyl, biphenyl,1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl,4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl,3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl,5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl,3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl,purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl groups.

The term “amine” or “amino,” as used herein, refers to an unsubstitutedor substituted moiety of the formula —NR^(a)R^(b), in which R^(a) andR^(b) are each independently hydrogen, alkyl, aryl, or heterocyclyl, orR^(a) and R^(b), taken together with the nitrogen atom to which they areattached, form a cyclic moiety having from 3 to 8 atoms in the ring.Thus, the term amino includes cyclic amino moieties such as piperidinylor pyrrolidinyl groups, unless otherwise stated. Thus, the term“alkylamino” as used herein means an alkyl group having an amino groupattached thereto. Suitable alkylamino groups include groups having 1 toabout 12 carbon atoms, preferably from 1 to about 6 carbon atoms. Theterm amino includes compounds or moieties in which a nitrogen atom iscovalently bonded to at least one carbon or heteroatom. The term“dialkylamino” includes groups wherein the nitrogen atom is bound to atleast two alkyl groups. The term “arylamino” and “diarylamino” includegroups wherein the nitrogen is bound to at least one or two aryl groups,respectively. The term “alkylarylamino” refers to an amino group whichis bound to at least one alkyl group and at least one aryl group. Theterm “alkaminoalkyl” refers to an alkyl, alkenyl, or alkynyl groupsubstituted with an alkylamino group. The term “amide” or“aminocarbonyl” includes compounds or moieties which contain a nitrogenatom which is bound to the carbon of a carbonyl or a thiocarbonyl group.

The term “alkylthio” refers to an alkyl group, having a sulfhydryl groupattached thereto. Suitable alkylthio groups include groups having 1 toabout 12 carbon atoms, preferably from 1 to about 6 carbon atoms.

The term “alkylcarboxyl” as used herein means an alkyl group having acarboxyl group attached thereto.

The term “alkoxy” as used herein means an alkyl group having an oxygenatom attached thereto. Representative alkoxy groups include groupshaving 1 to about 12 carbon atoms, preferably 1 to about 6 carbon atoms,e.g., methoxy, ethoxy, propoxy, tert-butoxy and the like. Examples ofalkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy,and pentoxy groups. The alkoxy groups can be substituted with groupssuch as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moieties. Examples of halogen substituted alkoxygroups include, but are not limited to, fluoromethoxy, difluoromethoxy,trifluoromethoxy, chloromethoxy, dichloromethoxy, trichloromethoxy,etc., as well as perhalogenated alkyloxy groups.

The term “acylamino” includes moieties wherein an amino moiety is bondedto an acyl group. For example, the acylamino group includesalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido groups.

The terms “alkoxyalkyl”, “alkylaminoalkyl” and “thioalkoxyalkyl” includealkyl groups, as described above, which further include oxygen, nitrogenor sulfur atoms replacing one or more carbons of the hydrocarbonbackbone.

The term “carbonyl” or “carboxy” includes compounds and moieties whichcontain a carbon connected with a double bond to an oxygen atom.Examples of moieties which contain a carbonyl include aldehydes,ketones, carboxylic acids, amides, esters, anhydrides, etc.

The term “ether” or “ethereal” includes compounds or moieties whichcontain an oxygen bonded to two carbon atoms. For example, an ether orethereal group includes “alkoxyalkyl” which refers to an alkyl, alkenyl,or alkynyl group substituted with an alkoxy group.

A “sulfonate” group is a —SO₃H or —SO₃ ⁻X⁺ group bonded to a carbonatom, where X⁺ is a cationic counter ion group. Similarly, a “sulfonicacid” compound has a —SO₃H or —SO₃ ⁻X⁺ group bonded to a carbon atom,where X⁺ is a cationic group. A “sulfate” as used herein is a —OSO₃H or—OSO₃ ⁻X⁺ group bonded to a carbon atom, and a “sulfuric acid” compoundhas a —SO₃H or —OSO₃ ⁻X⁺ group bonded to a carbon atom, where X⁺ is acationic group. According to the invention, a suitable cationic groupmay be a hydrogen atom. In certain cases, the cationic group mayactually be another group on the therapeutic compound that is positivelycharged at physiological pH, for example an amino group.

A “counter ion” is required to maintain electroneutrality. Examples ofanionic counter ions include halide, triflate, sulfate, nitrate,hydroxide, carbonate, bicarbonate, acetate, phosphate, oxalate, cyanide,alkylcarboxylate, N-hydroxysuccinimide, N-hydroxybenzotriazole,alkoxide, thioalkoxide, alkane sulfonyloxy, halogenated alkanesulfonyloxy, arylsulfonyloxy, bisulfate, oxalate, valerate, oleate,palmitate, stearate, laurate, borate, benzoate, lactate, citrate,maleate, fumarate, succinate, tartrate, naphthylate mesylate,glucoheptonate, or lactobionate. Compounds containing a cationic groupcovalently bonded to an anionic group may be referred to as an “internalsalt.”

The term “nitro” means —NO₂; the term “halogen” or “halogeno” or “halo”designates —F, —Cl, —Br or —I; the term “thiol,” “thio,” or “mercapto”means SH; and the term “hydroxyl” or “hydroxy” means —OH.

The term “acyl” refers to a carbonyl group that is attached through itscarbon atom to a hydrogen (i.e., a formyl), an aliphatic group (e.g.,acetyl), an aromatic group (e.g., benzoyl), and the like. The term“substituted acyl” includes acyl groups where one or more of thehydrogen atoms on one or more carbon atoms are replaced by, for example,an alkyl group, alkynyl group, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

Unless otherwise specified, the chemical moieties of the compounds ofthe invention, including those groups discussed above, may be“substituted or unsubstituted.” In some embodiments, the term“substituted” means that the moiety has substituents placed on themoiety other than hydrogen (i.e., in most cases, replacing a hydrogen),which allow the molecule to perform its intended function. Examples ofsubstituents include moieties selected from straight or branched alkyl(preferably C₁-C₅), cycloalkyl (preferably C₃-C₈), alkoxy (preferablyC₁-C₆), thioalkyl (preferably C₁-C₆), alkenyl (preferably C₂-C₆),alkynyl (preferably C₂-C₆), heterocyclic, carbocyclic, aryl (e.g.,phenyl), aryloxy (e.g., phenoxy), aralkyl (e.g., benzyl), aryloxyalkyl(e.g., phenyloxyalkyl), arylacetamidoyl, alkylaryl, heteroaralkyl,alkylcarbonyl and arylcarbonyl or other such acyl group,heteroarylcarbonyl, and heteroaryl groups, as well as (CR′R″)₀₋₃NR′R″(e.g., —NH₂), (CR′R″)₀₋₃CN (e.g., —CN), —NO₂, halogen (e.g., —F, —Cl,—Br, or —I), (CR′R″)₀₋₃C(halogen)₃ (e.g., —CF₃), (CR′R″)₀₋₃CH(halogen)₂,(CR′R″)₀₋₃CH₂(halogen), (CR′R″)₀₋₃CONR′R″, (CR′R″)₀₋₃(CNH)NR′R″,(CR′R″)₀₋₃S(O)₁₋₂NR′R″, (CR′R″)₀₋₃CHO, (CR′R″)₀₋₃O(CR′R″)₀₋₃H,(CR′R″)₀₋₃S(O)₀₋₃R′ (e.g., —SO₃H), (CR′R″)₀₋₃O(CR′R″)₀₋₃H (e.g.,—CH₂OCH₃ and —OCH₃), (CR′R″)₀₋₃S(CR′R″)₀₋₃H (e.g., —SH and —SCH₃),(CR′R″)₀₋₃OH (e.g., —OH), (CR′R″)₀₋₃COR′, (CR′R″)₀₋₃ (substituted orunsubstituted phenyl), (CR′R″)₀₋₃(C₃-C₈ cycloalkyl), (CR′R″)₀₋₃CO₂R′(e.g., —CO₂H), and (CR′R″)₀₋₃OR′ groups, wherein R′ and R″ are eachindependently hydrogen, a C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅ alkynyl, oraryl group; or the side chain of any naturally occurring amino acid.

In another embodiment, a substituent may be selected from straight orbranched alkyl (preferably C₁-C₅), cycloalkyl (preferably C₃-C₈), alkoxy(preferably C₁-C₆), thioalkyl (preferably C₁-C₆), alkenyl (preferablyC₂-C₆), alkynyl (preferably C₂-C₆), heterocyclic, carbocyclic, aryl(e.g., phenyl), aryloxy (e.g., phenoxy), aralkyl (e.g., benzyl),aryloxyalkyl (e.g., phenyloxyalkyl), arylacetamidoyl, alkylaryl,heteroaralkyl, alkylcarbonyl and arylcarbonyl or other such acyl group,heteroarylcarbonyl, or heteroaryl group, (CR′R″)₀₋₁₀NR′R″ (e.g., —NH₂),(CR′R″)₀₋₁₀CN (e.g., —CN), NO₂, halogen (e.g., F, Cl, Br, or I),(CR′R″)₀₋₁₀C(halogen)₃ (e.g., —CF₃), (CR′R″)₀₋₁₀CH(halogen)₂,(CR′R″)₀₋₁₀CH₂(halogen), (CR′R″)₀₋₁₀CONR′R″, (CR′R″)₀₋₁₀(CNH)NR′R″,(CR′R″)₀₋₁₀S(O)₁₋₂NR′R″, (CR′R″)₀₋₁₀CHO, (CR′R″)₀₋₁₀O(CR′R″)₀₋₁₀H,(CR′R″)₀₋₁₀S(O)₀₋₃R′ (e.g., —SO₃H), (CR′R″)₀₋₁₀O(CR′R″)₀₋₁₀H (e.g.,—CH₂OCH₃ and —OCH₃), (CR′R″)₀₋₁₀S(CR′R″)₀₋₃H (e.g., —SH and —SCH₃),(CR′R″)₀₋₁₀H (e.g., —OH), (CR′R″)₀₋₁₀COR′, (CR′R″)₀₋₁₀(substituted orunsubstituted phenyl), (CR′R″)₀₋₁₀(C₃-C₈ cycloalkyl), (CR′R″)₀₋₁₀CO₂R′(e.g., —CO₂H), or (CR′R″)₀₋₁₀OR′ group, or the side chain of anynaturally occurring amino acid; wherein R′and R″ are each independentlyhydrogen, a C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅ alkynyl, or aryl group, orR′ and R″ taken together are a benzylidene group or a —(CH₂)₂—O—(CH₂)₂—group.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance with thepermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc. As used herein, the term “substituted” ismeant to include all permissible substituents of organic compounds. In abroad aspect, the permissible substituents include acyclic and cyclic,branched and unbranched, carbocyclic and heterocyclic, aromatic andnonaromatic substituents of organic compounds. The permissiblesubstituents can be one or more.

In some embodiments, a “substituent” may be selected from the groupconsisting of, for example, halogeno, trifluoromethyl, nitro, cyano,C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkylcarbonyloxy,arylcarbonyloxy, C₁-C₆ alkoxycarbonyloxy, aryloxycarbonyloxy, C₁-C₆alkylcarbonyl, C₁-C₆ alkoxycarbonyl, C₁-C₆ alkoxy, C₁₋-C₆ alkylthio,arylthio, heterocyclyl, aralkyl, and aryl (including heteroaryl) groups.

In one embodiment, the invention pertains to compounds of Formula I:

wherein:

R¹ is a substituted or unsubstituted cycloalkyl, heterocyclic, aryl,arylcycloalkyl, bicyclic or tricyclic ring, a bicyclic or tricyclicfused ring group, or a substituted or unsubstituted C₂-C₁₀ alkyl group;

R² is selected from a group consisting of hydrogen, alkyl,mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl,triazolyl, imidazolyl, benzothiazolyl, and benzoimidazolyl;

Y is SO₃ ⁻X⁺, OSO₃ ⁻X⁺, or SSO₃ ⁻X⁺;

X⁺ is hydrogen, a cationic group, or ester-forming group; and

each of L¹ and L² is independently a substituted or unsubstituted C₁-C₅alkyl group or absent, or a pharmaceutically acceptable salt thereof,provided that when R₁ is alkyl, L¹ is absent.

In a further embodiment, the invention pertains to compounds of FormulaII:

wherein:

R¹ is a substituted or unsubstituted cyclic, bicyclic, tricyclic, orbenzoheterocyclic group or a substituted or unsubstituted C₂-C₁₀ alkylgroup;

R² is hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl,aryl, arylalkyl, thiazolyl, triazolyl, imidazolyl, benzothiazolyl,benzoimidazolyl, or linked to R¹ to form a heterocycle;

Y is SO₃ ⁻X⁺, OSO₃ ⁻X⁺, or SSO₃ ⁻X⁺;

X⁺ is hydrogen, a cationic group, or an ester forming moiety;

m is 0 or 1;

n is 1, 2, 3, or 4;

L is substituted or unsubstituted C₁-C₃ alkyl group or absent, or apharmaceutically acceptable salt thereof, provided that when R¹ isalkyl, L is absent.

In a further embodiment, R² is hydrogen. In another further embodiment,R¹ is straight chain alkyl, for example, ethyl, n-pentyl, n-heptyl, orn-octyl. In another embodiment, R¹ is t-butyl. In yet another alternateembodiment, R¹ is C₇-C₁₀ bicycloalkyl or tricycloalkyl, such as, forexample, tricyclo[3.3.1.0^(3,7)]decyl (or adamantyl),bicyclo[2.1.2]heptyl, or indolyl. In another alternate embodiment, R¹ istetrahydronaphthyl.

In one embodiment, L² is —(CH₂)₃—. In another further embodiment, L² is—(CH₂)₄— or —(CH₂)₅—. In yet another further embodiment, L₂ is —(CH₂)₂—.In yet another further embodiment, L² is substituted alkyl, e.g.,—CH₂—(CHOH)—CH₂—.

In another embodiment, L¹ is CH₂CH₂ or absent.

In a further embodiment, R¹ is branched alkyl, e.g., t-butyl. In anotherembodiment, R¹ is adamanyl. In another embodiment, R¹ is cyclic alkyl,e.g., cyclopropyl, cyclohexyl, cycloheptyl, cyclo-octyl, etc. Thecycloalkyl moieties may be substituted further, e.g., with additionalalkyl groups or other groups which allow the molecule to perform itsintended function. In another embodiment, R¹ is alkyl substituted with apropargyl moiety (e.g., HC≡C—). In another embodiment, R¹ is cyclohexylsubstituted with one or more methyl or propargyl groups.

In other embodiments, L¹ is a C₁-C₂ alkyl linker group (e.g., —CH(CH₃)—or —(CH₂)₂—. In a further embodiment, R¹ is phenyl. In certainembodiments, R¹ is substituted with a methoxy group. In otherembodiments, L¹ is C₃, e.g., —(CH₂)₃— or C(CH₃)₂—. In certainembodiments, L¹ is substituted, e.g., with an alkoxy, carboxylate(—COOH), benzyl, amido (—C═O—NH—), or ester (C═O—C—O) group. In certainembodiment, the ester group is a methyl, ethyl, propyl, butyl,cyclohexyl, or benzyl ester. In other embodiments, the ester group maybe propenyl. In other embodiments, L¹ is substituted with a carboxylategroup. In a further embodiment, R¹ is substituted with a substitutedamido group, wherein the amido group is substituted with an alkyl, e.g.,methyl, ethyl, propyl, butyl, pentyl, or hexyl group. In anotherembodiment, the alkyl R¹ group is a substituted with a —C═O—NH—OH,C═O—NH₂, or an amido group. In certain embodiments, the amido group issubstituted with an alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl,hexyl, cyclohexyl, etc.), a benzyl or an aryl group. In anotherembodiment, the amido group is substituted with a —CH(CH₂)₂ group. R¹itself may be substituted with a phenyl or may be branched or straightchain alkyl. In certain embodiments, R¹ may also be substituted with athioether moiety. Examples of thioethers include S-Me, S-Et, etc. Incertain embodiments, the alkyl R¹ moiety is substituted with both anaryl or a thioether moiety and an amido moiety. In other embodiments,the alkyl R¹ moiety may be substituted with both a thioether and acarboxylate moiety. In other embodiments, alkyl R¹ groups aresubstituted with hydroxyl. R¹ groups, e.g., alkyl R¹ groups, may also besubstituted with both thioether and hydroxyl groups. In otherembodiments, R¹ groups, e.g., alkyl R¹ groups are substituted with cyanogroups. Examples of R¹ groups including —CN moieties include —C(CH₃)₂CN,cyclohexyl substituted with one or more cyano groups, etc.

In other embodiments, alkyl R¹ groups are substituted with aryl groups.The aryl groups may be substituted phenyl, for example. The substitutedphenyl may be substituted with one or more substituents such as hydroxy,cyano and alkoxy. In other embodiments, alkyl R¹ groups are substitutedwith tetrazolyl or substituted or unsubstituted benzyl. In a furtherembodiment, L¹ is —C(CH₃)₂—(CH₂)—. In another embodiment, L¹ is—(C(CH₃)₂—CHOH—. In yet another embodiment, L¹ is —(C(CH₃)₂CH(OMe)-. Inanother embodiment, R¹ is substituted or unsubstituted phenyl. In afurther embodiment, R¹ is para-substituted phenyl. Examples ofsubstitutents include but are not limited to fluorine, chlorine,bromine, iodine, methyl, t-butyl, alkoxy, methoxy, etc. In otherembodiment, R¹ is substituted at the meta position. Examples ofsubstituents include methoxy, chloro, methyl, t-butyl, fluoro, alkyl,alkoxy, iodo, trifluoroalkyl, methoxy, etc. In another embodiment, R¹ isphenyl substituted in the ortho position, with similar substituents. Inanother embodiment, L¹ comprises a cycloalkyl moiety, e.g., cyclopentyl.In another embodiment, L¹ comprises an alkyenyl group and, optionally, asubstituted aryl group, with substitutents similar to those describedabout.

In certain embodiments, R¹ is cyclopropyl or cyclohexyl. In certainembodiments, the cyclopropyl or cyclohexyl group is substituted with anether group or an alkyl group. In certain further embodiments, the ethergroup is a benzyl ether group.

In another embodiment, wherein R¹ is alkyl, it is substituted withgroups such as phenyl, or hydroxy.

In other embodiments, the compound of the invention is selected from thegroup consisting of:

and pharmaceutically acceptable salts, esters, and prodrugs thereof.

In another embodiment, the invention pertains to compounds of FormulaIII:

wherein:

A is nitrogen or oxygen;

R¹¹ is hydrogen, salt-forming cation, ester forming group, —(CH₂)_(x)-Q,or when A is nitrogen, A and R¹¹ taken together may be the residue of anatural or unnatural amino acid or a salt or ester thereof;

Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl, orbenzoimidazolyl;

x is 0, 1, 2, 3, or 4;

n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

R³, R^(3a), R⁴, R^(4a), R⁵, R^(5a), R⁶, R^(6a), R⁷ and R^(7a) are eachindependently hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl,cycloalkyl, aryl, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, cyano,halogen, amino, tetrazolyl, or two R groups on adjacent ring atoms takentogether with the ring atoms form a double bond, provided that one ofR³, R^(3a), R⁴, R^(4a), R⁵, R^(5a), R⁶, R^(6a), R⁷ and R^(7a) is amoiety of the Formula IIIa:

wherein:

m is 0, 1, 2, 3, or 4;

R^(A), R^(B), R^(C), R^(D), and R^(E) are independently selected from agroup of hydrogen, halogen, hydroxyl, alkyl, alkoxyl, halogenated alkyl,mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, cyano, thiazolyl,triazolyl, imidazolyl, tetrazolyl, benzothiazolyl, and benzoimidazolyl;and pharmaceutically acceptable salts and esters thereof, provided thatsaid compound is not3-(4-phenyl-1,2,3,6-tetrahydro-1-pyridyl)-1-propanesulfonic acid.

In a further embodiment, n is 2, 3 or 4.

In another embodiment, R¹¹ is a salt-forming cation. Examples of saltforming cations include pharmaceutically acceptable salts describedherein as well as lithium, sodium, potassium, magnesium, calcium,barium, zinc, iron, and ammonium. In another embodiment, R¹¹ is anester-forming group. An ester-forming group includes groups which whenbound form an ester. Examples of such groups include substituted orunsubstituted alkyl, aryl, alkenyl, alkynyl, or cycloalkyl. In anotherembodiment, A is oxygen.

In another embodiment, R³ and R⁴ are taken together with the carbonatoms to which they are attached to form a double bond. In anotherembodiment, R^(A), R^(B), R^(C), R^(D), and R^(E) are each hydrogen.R^(A), R^(B), R^(D), and R^(E) are each hydrogen and R^(C) is a halogen,such as fluorine, chlorine, iodine, or bromine

In another embodiment, R³ or R^(5a) is a moiety of Formula IIIa.

In another embodiment, R⁴, R⁵, R⁶, and R⁷ are each hydrogen. In anotherfurther embodiment, R^(4a), R^(5a), R^(6a), and R^(7a) are eachhydrogen.

In another, R^(3a) is hydroxyl, cyano, acyl, or hydroxyl.

In another further embodiment, R¹¹ and A taken together are a natural orunnatural amino acid residue or a pharmaceutically acceptable salt orester thereof. Examples of amino acid residues include esters and saltsof phenylalanine and leucine.

In another embodiment, m is 0, 1, or 3.

Examples of compounds of Formula III include, but are not limited to:

and pharmaceutically acceptable salts, esters, and prodrugs thereof.

In another embodiment, the invention pertains to compounds of FormulaIV:

wherein:

A is nitrogen or oxygen;

R¹¹ is hydrogen, salt-forming cation, ester forming group, —(CH₂)_(x)-Q,or when A is nitrogen, A and R¹¹ taken together may be the residue of anatural or unnatural amino acid or a salt or ester thereof;

Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl, orbenzoimidazolyl;

x is 0, 1, 2, 3, or 4;

n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

R⁴, R^(4a), R⁵, R^(5a), R⁶, R^(6a), R⁷, and R^(7a) are eachindependently hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl,cycloalkyl, aryl, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, cyano,halogen, amino, tetrazolyl, R⁴ and R⁵ taken together, with the ringatoms they are attached to, form a double bond, or R⁶ and R⁷ takentogether, with the ring atoms they are attached to, form a double bond;

m is 0, 1, 2, 3, or 4;

R⁸, R⁹, R¹⁰, R¹¹, and R¹² are independently selected from a group ofhydrogen, halogen, hydroxyl, alkyl, alkoxyl, halogenated alkyl,mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, cyano, thiazolyl,triazolyl, imidazolyl, tetrazolyl, benzothiazolyl, and benzoimidazolyl,and pharmaceutically acceptable salts and esters thereof.

In another embodiment, R¹¹ is a salt-forming cation. Examples of saltforming cations include pharmaceutically acceptable salts describedherein as well as lithium, sodium, potassium, magnesium, calcium,barium, zinc, iron, and ammonium. In another embodiment, R¹¹ is anester-forming group. An ester-forming group includes groups which whenbound form an ester. Examples of such groups include substituted orunsubstituted alkyl, aryl, alkenyl, alkynyl, or cycloalkyl. In anotherembodiment, A is oxygen.

In another embodiment, m is 0 or 1. In another further embodiment, n is2, 3, or 4. In another further embodiment, R⁴, R⁵, R⁶ and R⁷ are eachhydrogen. R^(4a), R^(5a), R^(6a), and R^(7a) also may be hydrogen.Examples of R⁸, R⁹, R¹⁰, R¹¹, and R¹² include hydrogen. In anotherembodiment R⁸, R⁹, R¹¹, R¹² are each hydrogen, and R¹⁰ is a halogen,(e.g., fluorine, chlorine, bromine, or iodine), nitro, or alkyl (e.g.,methyl, ethyl, butyl).

In another embodiment, A-R¹¹ may be the residue of an amino acid, e.g.,a phenylalanine residue. In another embodiment, R⁹, R¹⁰, R¹¹ and R¹² areeach hydrogen, and R⁸ is not hydrogen, e.g., halogen, e.g., fluorine,bromine, chlorine, or iodine.

In another embodiment, the compound is:

and pharmaceutically acceptable salts, esters, and prodrugs thereof.

In another embodiment, the invention pertains to compounds of Formula V:

wherein:

A is nitrogen or oxygen;

R¹¹ is hydrogen, salt-forming cation, ester forming group, —(CH₂)_(x)-Q,or when A is nitrogen, A and R¹¹ taken together may be the residue of anatural or unnatural amino acid or a salt or ester thereof;

Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl, orbenzoimidazolyl;

x is 0, 1, 2, 3, or 4;

n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

aa is a natural or unnatural amino acid residue;

m is 0, 1, 2, or 3;

R¹⁴ is hydrogen or protecting group;

R¹⁵ is hydrogen, alkyl or aryl, and pharmaceutically acceptable salts,esters and prodrugs thereof.

In another embodiment, R¹¹ is a salt-forming cation. Examples of saltforming cations include pharmaceutically acceptable salts describedherein as well as lithium, sodium, potassium, magnesium, calcium,barium, zinc, iron, and ammonium. In another embodiment, R¹¹ is anester-forming group. An ester-forming group includes groups which whenbound form an ester. Examples of such groups include substituted orunsubstituted alkyl, aryl, alkenyl, alkynyl, or cycloalkyl. In anotherembodiment, A is oxygen.

In an embodiment, n is 2, 3 or 4. In certain embodiments, m is 0. Incertain embodiments, A-R¹¹ is a residue of a natural amino acid, or asalt or ester thereof. Examples of amino acid residues, include, but arenot limited to, leucine or phenylalanine residues, and pharmaceuticallyacceptable salts and esters thereof. Examples of possible esters includemethyl, ethyl, and t-butyl.

In another embodiment, m is 1. Examples of aa include natural andunnatural amino acid residues such as phenylalanine, glycine, andleucine.

In another embodiment, (aa)_(m) is a residue of phe-phe, or an esterthereof.

In certain embodiments, R¹⁵ is hydrogen or substituted alkyl, e.g.,arylalkyl. The term “unnatural amino acid” refers to any derivative of anatural amino acid including D forms, and α- and β-amino acidderivatives. It is noted that certain amino acids, e.g., hydroxyproline,that are classified as a non-natural amino acid herein, may be found innature within a certain organism or a particular protein. Amino acidswith many different protecting groups appropriate for immediate use inthe solid phase synthesis of peptides are commercially available. Inaddition to the twenty most common naturally occurring amino acids, thefollowing examples of non-natural amino acids and amino acid derivativesmay be used according to the invention (common abbreviations inparentheses): β-alanine (β-ALA), γ-aminobutyric acid (GABA),2-aminobutyric acid (2-Abu), α,β-dehydro-2-aminobutyric acid (8-AU),1-aminocyclopropane-1-carboxylic acid (ACPC), aminoisobutyric acid(Aib), 2-amino-thiazoline-4-carboxylic acid, 5-aminovaleric acid(5-Ava), 6-aminohexanoic acid (6-Ahx), 8-aminooctanoic acid (8-Aoc),11-aminoundecanoic acid (11-Aun), 12-aminododecanoic acid (12-Ado),2-aminobenzoic acid (2-Abz), 3-aminobenzoic acid (3-Abz), 4-aminobenzoicacid(4-Abz), 4-amino-3-hydroxy-6-methylheptanoic acid (Statine, Sta),aminooxyacetic acid (Aoa), 2-aminotetraline-2-carboxylic acid (ATC),4-amino-5-cyclohexyl-3-hydroxypentanoic acid (ACHPA),para-aminophenylalanine (4-NH₂-Phe), biphenylalanine (Bip),para-bromophenylalanine (4-Br-Phe),ortho-chlorophenylalanine](2-Cl-Phe), meta-chlorophenylalanine(3-Cl-Phe), para-chlorophenylalanine (4-Cl-Phe), meta-chlorotyrosine(3-Cl-Tyr), para-benzoylphenylalanine (Bpa), tert-butylglycine (TLG),cyclohexylalanine (Cha), cyclohexylglycine (Chg), 2,3-diaminopropionicacid (Dpr), 2,4-diaminobutyric acid (Dbu), 3,4-dichlorophenylalanine(3,4-Cl₂-Phe), 3,4-difluororphenylalanine (3,4-F₂-Phe),3,5-diiodotyrosine (3,5-I₂-Tyr), ortho-fluorophenylalanine (2-F-Phe),meta-fluorophenylalanine (3-F-Phe), para-fluorophenylalanine (4-F-Phe),meta-fluorotyrosine (3-F-Tyr), homoserine (Hse), homophenylalanine(Hfe), homotyrosine (Htyr), 5-hydroxytryptophan (5-OH-Trp),hydroxyproline (Hyp), para-iodophenylalanine (4-I-Phe), 3-iodotyrosine(3-I-Tyr), indoline-2-carboxylic acid (Idc), isonipecotic acid (Inp),meta-methyltyrosine (3-Me-Tyr), 1-naphthylalanine (1-Nal),2-naphthylalanine (2-Nal), para-nitrophenylalanine (4-NO₂-Phe),3-nitrotyrosine (3-NO₂-Tyr), norleucine (Nle), norvaline (Nva),ornithine (Orn), ortho-phosphotyrosine (H₂PO₃-Tyr),octahydroindole-2-carboxylic acid (Oic), penicillamine (Pen),pentafluorophenylalanine (F₅-Phe), phenylglycine (Phg), pipecolic acid(Pip), propargylglycine (Pra), pyroglutamic acid (PGLU), sarcosine(Sar), tetrahydroisoquinoline-3-carboxylic acid (Tic), thienylalanine,and thiazolidine-4-carboxylic acid (thioproline, Th). Additionally,N-alkylated amino acids may be used, as well as amino acids havingamine-containing side chains (such as Lys and Orn) in which the aminehas been acylated or alkylated.

Examples of compounds of the invention include, but are not limited to:

and pharmaceutically acceptable salts, esters, and prodrugs thereof.

In another embodiment, the invention pertains, at least in part, tocompounds of Formula VI:

wherein:

n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

A is oxygen or nitrogen;

R¹¹ is hydrogen, salt-forming cation, ester forming group, —(CH₂)_(x)-Q,or when A is nitrogen, A and R¹¹ taken together may be the residue of anatural or unnatural amino acid or a salt or ester thereof;

Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl, orbenzoimidazolyl;

x is 0, 1, 2, 3, or 4;

R¹⁹ is hydrogen, alkyl or aryl;

Y¹ is oxygen, sulfur, or nitrogen;

Y² is carbon, nitrogen, or oxygen;

R²⁰ is hydrogen, alkyl, amino, mercaptoalkyl, alkenyl, alkynyl,cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, tetrazolyl,imidazolyl, benzothiazolyl, or benzoimidazolyl;

R²¹ is hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl,aryl, arylalkyl, thiazolyl, triazolyl, tetrazolyl, imidazolyl,benzothiazolyl, benzoimidazolyl, or absent if Y² is oxygen;

R²² is hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl,aryl, arylalkyl, thiazolyl, triazolyl, tetrazolyl, imidazolyl,benzothiazolyl, benzoimidazolyl; or R²² is hydrogen, hydroxyl, alkoxy oraryloxy if Y¹ is nitrogen; or R²² is absent if Y¹ is oxygen or sulfur;or R²² and R²¹ may be linked to form a cyclic moiety if Y¹ is nitrogen;

R²³ is hydrogen, alkyl, amino, mercaptoalkyl, alkenyl, alkynyl,cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, tetrazolyl,imidazolyl, benzothiazolyl, or benzoimidazolyl, or absent if Y² isnitrogen or oxygen;

or pharmaceutically acceptable salts thereof.

In another embodiment, R¹¹ is a salt-forming cation. Examples of saltforming cations include pharmaceutically acceptable salts describedherein as well as lithium, sodium, potassium, magnesium, calcium,barium, zinc, iron, and ammonium. In a further embodiment, the salt is asodium salt. In a further, embodiment, A is oxygen.

In another embodiment, Y¹ is oxygen or sulfur, and R²² is absent.

In another embodiment, Y² is oxygen and R²¹ is absent. Examples of R²⁰include benzyl, aryl (e.g., phenyl), alkyl, cycloalkyl (e.g.,adamantyl), etc. In other embodiment, Y² is nitrogen and R²¹ ishydrogen. In other embodiment, R²¹ is benzyl. In another furtherembodiment, R²⁰ and R²¹ are linked to form a pyridyl ring. In anotherembodiment, Y¹ is sulfur.

Examples of compounds of the invention, include

and pharmaceutically acceptable salts, esters, and prodrugs thereof.

In another embodiment, the invention pertains to compounds of FormulaVII:

wherein:

n is 2, 3, or 4;

A is oxygen or nitrogen;

R¹¹ is hydrogen, salt-forming cation, ester forming group, —(CH₂)_(x)-Q,or when A is nitrogen, A and R¹¹ taken together may be the residue of anatural or unnatural amino acid or a salt or ester thereof;

Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl, orbenzoimidazolyl;

x is 0, 1, 2, 3, or 4;

G is a direct bond or oxygen, nitrogen, or sulfur;

z is 0, 1, 2, 3, 4, or 5;

m is 0 or 1;

R²⁴ is selected from a group consisting of hydrogen, alkyl,mercaptoalkyl, alkenyl, alkynyl, aroyl, alkylcarbonyl,aminoalkylcarbonyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl,imidazolyl, benzothiazolyl, and benzoimidazolyl;

each R²⁵ is independently selected from hydrogen, halogen, cyano,hydroxyl, alkoxy, thiol, amino, nitro, alkyl, aryl, carbocyclic, orheterocyclic, and pharmaceutically acceptable salts, esters, andprodrugs thereof.

In one embodiment, R¹¹ is hydrogen. In another, A is oxygen. Forexample, n may be 3 and m may be 1. In other embodiments, R²⁴ ishydrogen or benzyl.

In certain embodiments, z is 0, 2, or 3. In others, R²⁵ is hydroxyl oralkoxy, e.g., methoxy, ethoxy, etc. In certain embodiments, two or moreR²⁵ substituents can be linked to form a fused ring (e.g., to form amethylendioxyphenyl moiety).

Examples of compounds of the invention include:

and pharmaceutically acceptable salts, esters, and prodrugs thereof.

Other compounds of the invention include

and pharmaceutically acceptable salts, esters, and prodrugs thereof.

The invention pertains to both salt forms and acid/base forms of thecompounds of the invention. For example, the invention pertains not onlyto the particular salt forms of compounds shown herein as salts, butalso the invention includes other pharmaceutically acceptable salts, andthe acid and/or base form of the compound. The invention also pertainsto salt forms of compounds shown herein.

Compounds of the invention are also shown in Table 2 below.

TABLE 2 ID Structure/Name of Compound B

C

D

E

F

G

H

I

J

K

L

M

N

P

Q

R

S

X

Y

Z

AA

AB

AC

AD

AE

AF

AG

AH

AI

AJ

AK

AL

AM

AU

AV

AW

AX

AY

AZ

BA

BB

BC

BW

BX

BY

BZ

CC

CD

CE

CG

CH

CI

CJ

CK

CL

CM

CN

CO

CV

CY

DC

DD

DE

DG

DH

DI

DJ

DK

DL

DM

DN

DO

DP

DQ

DR

DS

DT

DU

DV

DW

DX

DY

DZ

EA

EB

EC

ED

EE

EF

EG

EH

EI

EJ

EK

EL

EN

EO

EP

EQ

ER

ES

ET

EV

EW

EY

EZ

FA

FH

FL

FM

FN

FO

FP

FQ

FR

FS

FT

FU

FV

FW

FX

FY

FZ

GA

GB

GC

GD

GE

GF

GH

GI

GJ

GK

GL

GM

GN

GO

GP

GQ

GR

GS

GT

GU

GZ

HA

HB

HC

HD

HE

HF

HG

HI

HJ

HK

HL

HM

HN

HO

HP

HQ

HR

HS

HT

HU

HV

HW

HX

HY

HZ

IA

IB

IC

ID

IE

IF

IG

IH

II

IJ

IK

IL

IM

IN

IO

IP

IR

IS

IT

IU

IV

IW

IX

IY

IZ

JA

JB

JC

JD

JE

JF

JG

JH

JI

JJ

JK

JL

JM

JN

JO

JP

JQ

JR

JS

JT

JU

JV

JW

JX

JY

JZ

KA

KB

KH

KI

KJ

KK

KL

KM

KN

KP

KQ

KR

KS

KT

KV

KW

KX

KY

LA

LC

LD

LE

LF

LG

LH

LI

LJ

LK

LL

LM

LN

LO

LP

LQ

NE

NG

NH

NI

NJ

NK

NL

It should be noted that in the above table and throughout theapplication when an atom is shown without hydrogens, but hydrogens arerequired or chemically necessary to form a stable compound, hydrogensshould be inferred to be part of the compound.

In one embodiment, the invention does not pertain to the compoundsdescribed in WO 00/64420 and WO 96/28187. In this embodiment, theinvention does not pertain to methods of using the compounds describedin WO 00/64420 and WO 96/28187 for the treatment of diseases ordisorders described therein. In a further embodiment, the inventionpertains to methods of using the compounds described in WO 00/64420 andWO 96/28187 for methods described in this application, which are notdescribed in WO 00/64420 and WO 96/28187. Both of WO 00/64420 and WO96/28187 are incorporated by reference herein in their entirety.

In another embodiment, the invention pertains to methods of theinvention which use and pharmaceutical compositions comprising, thecompounds of Table 2A. In another embodiment, the compounds of theinvention do not include the compounds of Table 2A.

TABLE 2A

BzNHCH₂CH₂CH₂SO₃Na

AcNHCH₂CH₂CH₂SO₃Na

CH₃(CH₂)₈NH(CH₂)₃SO₃H

CH₃(CH₂)₉NH(CH₂)₃SO₃H

CH₃(CH₂)₁₁NH(CH₂)₃SO₃H CH₃(CH₂)₁₀NH(CH₂)₃SO₃H CH₃(CH₂)₁₂NH(CH₂)₃SO₃H

HOCH₂CH₂CH₂NHCH₂CH₂CH₂SO₃] CH₃(CH₂)₁₃NH(CH₂)₃SO₃H CH₃(CH₂)₁₅NH(CH₂)₃SO₃H

CH₃(CH₂)₁₇NHCH₂CH₂CH₂SO₃H

CH₃(CH₂)₁₃N⁺(CH₃)₂[(CH₂)₃SO₃ ⁻]

NaO₃SOCH₂(CH₂)₃CH₂OSO₃Na

HOCH₂CH₂CH₂CH₂SO₃Na (NaO₃SCH₂CH₂CH₂CH₂)₂O

CH₃C(CH₂OSO₃Na)₃ NH₂CH₂CH₂CH₂SO₃Na HC(CH₂OSO₃Na)₃ NH₂C(CH₂OSO₃Na)₃NH₂CH₂CH₂OSO₃H NaO₃SNHCH₂CH₂OSO₃Na

NaO₃SNHCH₂CH₂CH₂OSO₃Na HN(CH₂CH₂OSO₃Na)₂ H₂NCH₂CH₂CH₂OSO₃NaNaO₃SN(CH₂CH₂OSO₃Na)₂ H₂NCH₂CH₂SO₃H NaO₃SOCH₂CH₂CH₂SO₃Na

CH₃CH₂CH₂CH₂SO₃Na CH₃(CH₂)₈CH₂SO₃Na

CH₃CH₂CH₂SO₃Na CH₃CH₂SO₃Na CH₃CH₂CH₂CH₂CH₂SO₃Na

It should be understood that the use of any of the compounds describedherein or in the applications identified in “The Related Applications”Section is within the scope of the present invention and is intended tobe encompassed by the present invention and each of the applications areexpressly incorporated herein at least for these purposes, and arefurthermore expressly incorporated for all other purposes.

Subjects and Patient Populations

The term “subject” includes living organisms in which amyloidosis canoccur, or which are susceptible to amyloid diseases, e.g., Alzheimer'sdisease, Down's syndrome, CAA, dialysis-related (β₂M) amyloidosis,secondary (AA) amyloidosis, primary (AL) amyloidosis, hereditaryamyloidosis, diabetes, etc. Examples of subjects include humans,chickens, ducks, peking ducks, geese, monkeys, deer, cows, rabbits,sheep, goats, dogs, cats, mice, rats, and transgenic species thereof.Administration of the compositions of the present invention to a subjectto be treated can be carried out using known procedures, at dosages andfor periods of time effective to modulate amyloid aggregation oramyloid-induced toxicity in the subject as further described herein. Aneffective amount of the therapeutic compound necessary to achieve atherapeutic effect may vary according to factors such as the amount ofamyloid already deposited at the clinical site in the subject, the age,sex, and weight of the subject, and the ability of the therapeuticcompound to modulate amyloid aggregation in the subject. Dosage regimenscan be adjusted to provide the optimum therapeutic response. Forexample, several divided doses may be administered daily or the dose maybe proportionally reduced as indicated by the exigencies of thetherapeutic situation.

In certain embodiments of the invention, the subject is in need oftreatment by the methods of the invention, and is selected for treatmentbased on this need. A subject in need of treatment is art-recognized,and includes subjects that have been identified as having a disease ordisorder related to amyloid-deposition or amyloidosis, has a symptom ofsuch a disease or disorder, or is at risk of such a disease or disorder,and would be expected, based on diagnosis, e.g., medical diagnosis, tobenefit from treatment (e.g., curing, healing, preventing, alleviating,relieving, altering, remedying, ameliorating, improving, or affectingthe disease or disorder, the symptom of the disease or disorder, or therisk of the disease or disorder).

In an exemplary aspect of the invention, the subject is a human. Forexample, the subject may be a human over 30 years old, human over 40years old, a human over 50 years old, a human over 60 years old, a humanover 70 years old, a human over 80 years old, a human over 85 years old,a human over 90 years old, or a human over 95 years old. The subject maybe a female human, including a postmenopausal female human, who may beon hormone (estrogen) replacement therapy. The subject may also be amale human. In another embodiment, the subject is under 40 years old.

A subject may be a human at risk for Alzheimer's disease, e.g., beingover the age of 40 or having a predisposition for Alzheimer's diseaseAlzheimer's disease predisposing factors identified or proposed in thescientific literature include, among others, a genotype predisposing asubject to Alzheimer's disease; environmental factors predisposing asubject to Alzheimer's disease; past history of infection by viral andbacterial agents predisposing a subject to Alzheimer's disease; andvascular factors predisposing a subject to Alzheimer's disease. Asubject may also have one or more risk factors for cardiovasculardisease (e.g., atherosclerosis of the coronary arteries, anginapectoris, and myocardial infarction) or cerebrovascular disease (e.g.,atherosclerosis of the intracranial or extracranial arteries, stroke,syncope, and transient ischemic attacks), such as hypercholesterolemia,hypertension, diabetes, cigarette smoking, familial or previous historyof coronary artery disease, cerebrovascular disease, and cardiovasculardisease. Hypercholesterolemia typically is defined as a serum totalcholesterol concentration of greater than about 5.2 mmol/L (about 200mg/dL).

Several genotypes are believed to predispose a subject to Alzheimer'sdisease. These include the genotypes such as presenilin-1, presenilin-2,and amyloid precursor protein (APP) missense mutations associated withfamilial Alzheimer's disease, and α-2-macroglobulin and LRP-1 genotypes,which are thought to increase the risk of acquiring sporadic(late-onset) Alzheimer's disease. E.van Uden, et al., J. Neurosci.22(21), 9298-304 (2002); J. J. Goto, et al., J. Mol. Neurosci. 19(1-2),37-41 (2002). Another genetic risk factor for the development ofAlzheimer's disease are variants of ApoE, the gene that encodesapolipoprotein E (particularly the apoE4 genotype), a constituent of thelow-density lipoprotein particle. W J Strittmatter, et al., Annu. Rev.Neurosci. 19, 53-77 (1996). The molecular mechanisms by which thevarious ApoE alleles alter the likelihood of developing Alzheimer'sdisease are unknown, however the role of ApoE in cholesterol metabolismis consistent with the growing body of evidence linking cholesterolmetabolism to Alzheimer's disease. For example, chronic use ofcholesterol-lowering drugs such as statins has recently been associatedwith a lower incidence of Alzheimer's disease, and cholesterol-loweringdrugs have been shown to reduce pathology in APP transgenic mice. Theseand other studies suggest that cholesterol may affect APP processing.ApoE4 has been suggested to alter Aβ trafficking (in and out of thebrain), and favor retention of Aβ in the brain. ApoE4 has also beensuggested to favor APP processing toward Aβ formation. Environmentalfactors have been proposed as predisposing a subject to Alzheimer'sdisease, including exposure to aluminum, although the epidemiologicalevidence is ambiguous. In addition, prior infection by certain viral orbacterial agents may predispose a subject to Alzheimer's disease,including the herpes simplex virus and chlamydia pneumoniae. Finally,other predisposing factors for Alzheimer's disease can include riskfactors for cardiovascular or cerebrovascular disease, includingcigarette smoking, hypertension and diabetes. “At risk for Alzheimer'sdisease” also encompasses any other predisposing factors not listedabove or as yet identified and includes an increased risk forAlzheimer's disease caused by head injury, medications, diet, orlifestyle.

The methods of the present invention can be used for one or more of thefollowing: to prevent Alzheimer's disease, to treat Alzheimer's disease,or ameliorate symptoms of Alzheimer's disease, or to regulate productionof or levels of amyloid β (Aβ) peptides. In an embodiment, the humancarries one or more mutations in the genes that encode β-amyloidprecursor protein, presenilin-1 or presenilin-2. In another embodiment,the human carries the Apolipoprotein ε4 gene. In another embodiment, thehuman has a family history of Alzheimer's Disease or a dementia illness.In another embodiment, the human has trisomy 21 (Down's Syndrome). Inanother embodiment, the subject has a normal or low serum total bloodcholesterol level. In another embodiment, the serum total bloodcholesterol level is less than about 200 mg/dL, or less than about 180,and it can range from about 150 to about 200 mg/dL. In anotherembodiment, the total LDL cholesterol level is less than about 100mg/dL, or less than about 90 mg/dL and can range from about 30 to about100 mg/dL. Methods of measuring serum total blood cholesterol and totalLDL cholesterol are well known to those skilled in the art and forexample include those disclosed in WO 99/38498 at p. 11, incorporated byreference herein. Methods of determining levels of other sterols inserum are disclosed in H. Gylling, et al., “Serum Sterols During StanolEster Feeding in a Mildly Hypercholesterolemic Population”, J. LipidRes. 40: 593-600 (1999).

In another embodiment, the subject has an elevated serum total bloodcholesterol level. In another embodiment, the serum total cholesterollevel is at least about 200 mg/dL, or at least about 220 mg/dL and canrange from about 200 to about 1000 mg/dL. In another embodiment, thesubject has an elevated total LDL cholesterol level. In anotherembodiment, the total LDL cholesterol level is greater than about 100mg/dL, or even greater than about 110 mg/dL and can range from about 100to about 1000 mg/dL.

In another embodiment, the human is at least about 40 years of age. Inanother embodiment, the human is at least about 60 years of age. Inanother embodiment, the human is at least about 70 years of age. Inanother embodiment, the human is at least about 80 years of age. Inanother embodiment, the human is at least about 85 years of age. In oneembodiment, the human is between about 60 and about 100 years of age.

In still a further embodiment, the subject is shown to be at risk by adiagnostic brain imaging technique, for example, one that measures brainactivity, plaque deposition, or brain atrophy.

In still a further embodiment, the subject is shown to be at risk by acognitive test such as Clinical Dementia Rating (“CDR”), Alzheimer'sDisease Assessment Scale-Cognition (“ADAS-Cog”), Disability Assessmentfor Dementia (“DAD”) or Mini-Mental State Examination (“MMSE”). Thesubject may exhibit a below average score on a cognitive test, ascompared to a historical control of similar age and educationalbackground. The subject may also exhibit a reduction in score ascompared to previous scores of the subject on the same or similarcognition tests.

In determining the CDR, a subject is typically assessed and rated ineach of six cognitive and behavioural categories: memory, orientation,judgement and problem solving, community affairs, home and hobbies, andpersonal care. The assessment may include historical informationprovided by the subject, or preferably, a corroborator who knows thesubject well. The subject is assessed and rated in each of these areasand the overall rating, (0, 0.5, 1.0, 2.0 or 3.0) determined. A ratingof 0 is considered normal. A rating of 1.0 is considered to correspondto mild dementia. A subject with a CDR of 0.5 is characterized by mildconsistent forgetfulness, partial recollection of events and “benign”forgetfulness. In one embodiment the subject is assessed with a ratingon the CDR of above 0, of above about 0.5, of above about 1.0, of aboveabout 1.5, of above about 2.0, of above about 2.5, or at about 3.0.

Another test is the Mini-Mental State Examination (MMSE), as describedby Folstein “Mini-mental state. A practical method for grading thecognitive state of patients for the clinician.” J. Psychiatr. Res.12:189-198, 1975. The MMSE evaluates the presence of global intellectualdeterioration. See also Folstein “Differential diagnosis of dementia.The clinical process.” Psychiatr Clin North Am. 20:45-57, 1997. The MMSEis a means to evaluate the onset of dementia and the presence of globalintellectual deterioration, as seen in Alzheimer's disease andmulti-infart dementia. The MMSE is scored from 1 to 30. The MMSE doesnot evaluate basic cognitive potential, as, for example, the so-calledIQ test. Instead, it tests intellectual skills. A person of “normal”intellectual capabilities will score a “30” on the MMSE objective test(however, a person with a MMSE score of 30 could also score well below“normal” on an IQ test). See, e.g., Kaufer, J. Neuropsychiatry Clin.Neurosci. 10:55-63, 1998; Becke, Alzheimer Dis Assoc Disord. 12:54-57,1998; Ellis, Arch. Neurol. 55:360-365, 1998; Magni, Int. Psychogeriatr.8:127-134, 1996; Monsch, Acta Neurol. Scand. 92:145-150, 1995. In oneembodiment, the subject scores below 30 at least once on the MMSE. Inanother embodiment, the subject scores below about 28, below about 26,below about 24, below about 22, below about 20, below about 18, belowabout 16, below about 14, below about 12, below about 10, below about 8,below about 6, below about 4, below about 2, or below about 1.

The Disability Assessment for Dementia (“DAD”) scale has been developedto measure a patient's ability to perform the activities of daily living(Gélinas I et al. Development of a Functional Measure for Persons withAlzheimer's Disease: The Disability Assessment for Dementia. Am. J.Occupational Therapy. 1999; 53: 471-481). Activities of daily living maybe assessed according to self care (i.e., dressing and personal hygiene)and instrumental activities (e.g., housework, cooking, and usinghousehold devices). The objectives of the DAD scale includequantitatively measuring functional abilities in activities of dailyliving in individuals with cognitive impairments and to help delineateareas of cognitive deficits that may impair performance in activities ofdaily living. The DAD is administered through an interview with thecaregiver. It measures actual performance in activities of daily livingof the individual as observed over a 2 week period prior to theinterview. The scale assesses the following domains of activities:hygiene, dressing, telephoning, continence, eating, meal preparation,outing activities, finance and correspondence, medication use, leisureand housework. A total score is obtained by adding the rating for eachquestion and converting this total score out of 100. Higher scoresrepresent less disability in ADL while lower scores indicate moredysfunction. In one embodiment, the subject scores below 100 at leastonce on the DAD. In another embodiment, the subject scores below about95, below about 90, below about 85, below about 80, below about 75,below about 70, below about 65, below about 60, below about 55, belowabout 50, below about 45, below about 40, below about 30, below about20, or below about 10.

Another means to evaluate cognition, particularly Alzheimer's disease,is the Alzheimer's Disease Assessment Scale (ADAS-Cog), or a variationtermed the Standardized Alzheimer's Disease Assessment Scale (SADAS). Itis commonly used as an efficacy measure in clinical drug trials ofAlzheimer's disease and related disorders characterized by cognitivedecline. SADAS and ADAS-Cog were not designed to diagnose Alzheimer'sdisease; they are useful in characterizing symptoms of dementia and area relatively sensitive indicator of dementia progression. (See, e.g.,Doraiswamy, Neurology 48:1511-1517, 1997; and Standish, J. Am. Geriatr.Soc. 44:712-716, 1996.) Annual deterioration in untreated Alzheimer'sdisease patients is approximately 8 points per year (See, e.g., Raskind,M. Prim. Care Companion J Clin Psychiatry 2000 August; 2(4):134-138).

The ADAS-cog is designed to measure, with the use of questionnaires, theprogression and the severity of cognitive decline as seen in AD on a70-point scale. The ADAS-cog scale quantifies the number of wronganswers. Consequently, a high score on the scale indicates a more severecase of cognitive decline. In one embodiment, a subject exhibits a scoreof greater than 0, greater than about 5, greater than about 10, greaterthan about 15, greater than about 20, greater than about 25, greaterthan about 30, greater than about 35, greater than about 40, greaterthan about 45, greater than about 50, greater than about 55, greaterthan about 60, greater than about 65, greater than about 68, or about70. In another embodiment, the subject exhibits no symptoms ofAlzheimer's Disease. In another embodiment, the subject is a human whois at least 40 years of age and exhibits no symptoms of Alzheimer'sDisease. In another embodiment, the subject is a human who is at least40 years of age and exhibits one or more symptoms of Alzheimer'sDisease.

In another embodiment, the subject has Mild Cognitive Impairment. In afurther embodiment, the subject has a CDR rating of about 0.5. Inanother embodiment, the subject has early Alzheimer's disease. Inanother embodiment, the subject has cerebral amyloid angiopathy.

By using the methods of the present invention, the levels of amyloid βpeptides in a subject's plasma or cerebrospinal fluid (CSF) can bereduced from levels prior to treatment from about 10 to about 100percent, or even about 50 to about 100 percent.

In an alternative embodiment, the subject can have an elevated level ofamyloid Aβ₄₀ and Aβ₄₂ peptide in the blood and CSF prior to treatment,according to the present methods, of greater than about 10 pg/mL, orgreater than about 20 pg/mL, or greater than about 35 pg/mL, or evengreater than about 40 pg/mL. In another embodiment, the elevated levelof amyloid Aβ₄₂ peptide can range from about 30 pg/mL to about 200pg/mL, or even to about 500 pg/mL. One skilled in the art wouldunderstand that as Alzheimer's disease progresses, the measurable levelsof amyloid β peptide in the CSF may decrease from elevated levelspresent before onset of the disease. This effect is attributed toincreased deposition, i.e., trapping of Aβ peptide in the brain insteadof normal clearance from the brain into the CSF.

In an alternative embodiment, the subject can have an elevated level ofamyloid Aβ₄₀ peptide in the blood and CSF prior to treatment, accordingto the present methods, of greater than about 5 pg Aβ₄₂/mL or greaterthan about 50 pg Aβ₄₀/mL, or greater than about 400 pg/mL. In anotherembodiment, the elevated level of amyloid Aβ₄₀ peptide can range fromabout 200 pg/mL to about 800 pg/mL, to even about 1000 pg/mL.

In another embodiment, the subject can have an elevated level of amyloidAβ₄₂ peptide in the CSF prior to treatment, according to the presentmethods, of greater than about 10 pg/mL, or greater than about 10 pg/mL,or greater than about 200 pg/mL, or greater than about 500 pg/mL. Inanother embodiment, the level of amyloid β peptide can range from about10 pg/mL to about 1,000 pg/mL, or even about 100 pg/mL to about 1,000pg/mL.

In another embodiment, the subject can have an elevated level of amyloidAβ₄₀ peptide in the CSF prior to treatment according to the presentmethods of greater than about pg/mL, or greater than about 50 pg/mL, oreven greater than about 100 pg/mL. In another embodiment, the level ofamyloid β peptide can range from about 10 pg/mL to about 1,000 pg/mL.

The amount of amyloid β peptide in the brain, CSF, blood, or plasma of asubject can be evaluated by enzyme-linked immunosorbent assay (“ELISA”)or quantitative immunoblotting test methods or by quantitative SELDI-TOFwhich are well known to those skilled in the art, such as is disclosedby Zhang, et al., J. Biol. Chem. 274, 8966-72 (1999) and Zhang, et al.,Biochemistry 40, 5049-55 (2001). See also, A. K. Vehmas, et al., DNACell Biol. 20(11), 713-21 (2001), P. Lewczuk, et al., Rapid Commun. MassSpectrom. 17(12), 1291-96 (2003); B. M. Austen, et al., J. Peptide Sci.6, 459-69 (2000); and H. Davies, et al., BioTechniques 27, 1258-62(1999). These tests are performed on samples of the brain or blood whichhave been prepared in a manner well known to one skilled in the art.Another example of a useful method for measuring levels of amyloid βpeptides is by Europium immunoassay (EIA). See, e.g., WO 99/38498 at p.11.

The methods of the invention may be applied as a therapy for a subjecthaving Alzheimer's disease or a dementia, or the methods of theinvention may be applied as a prophylaxis against Alzheimer's disease ordementia for subject with such a predisposition, as in a subject, e.g.,with a genomic mutation in the APP gene, the ApoE gene, or a presenilingene. The subject may have (or may be predisposed to developing or maybe suspected of having) vascular dementia, or senile dementia, MildCognitive Impairment, or early Alzheimer's disease. In addition toAlzheimer's disease, the subject may have another amyloid-relateddisease such as cerebral amyloid angiopathy, or the subject may haveamyloid deposits, especially amyloid-β amyloid deposits in the brain.

Treatment of Amyloid-Related Diseases

The present invention pertains to methods of using the compounds andpharmaceutical compositions thereof in the treatment and prevention ofamyloid-related diseases. The pharmaceutical compositions of theinvention may be administered therapeutically or prophylactically totreat diseases associated with amyloid (e.g., AL amyloid protein (λ orκ-chain related, e.g., amyloid λ, amyloid κ, amyloid κIV, amyloid λVI,amyloid γ, amyloid γ1), Aβ, IAPP, β₂M, AA, or AH amyloid protein) fibrilformation, aggregation or deposition.

The pharmaceutical compositions of the invention may act to amelioratethe course of an amyloid-related disease using any of the followingmechanisms (this list is meant to be illustrative and not limiting):slowing the rate of amyloid fibril formation or deposition; lesseningthe degree of amyloid deposition; inhibiting, reducing, or preventingamyloid fibril formation; inhibiting neurodegeneration or cellulartoxicity induced by amyloid; inhibiting amyloid induced inflammation;enhancing the clearance of amyloid from the brain; enhancing degradationof Aβ in the brain; or favoring clearance of amyloid protein prior toits organization in fibrils.

“Modulation” of amyloid deposition includes both inhibition, as definedabove, and enhancement of amyloid deposition or fibril formation. Theterm “modulating” is intended, therefore, to encompass prevention orstopping of amyloid formation or accumulation, inhibition or slowingdown of further amyloid formation or accumulation in a subject withongoing amyloidosis, e.g., already having amyloid deposition, andreducing or reversing of amyloid formation or accumulation in a subjectwith ongoing amyloidosis; and enhancing amyloid deposition, e.g.,increasing the rate or amount of amyloid deposition in vivo or in vitro.Amyloid-enhancing compounds may be useful in animal models ofamyloidosis, for example, to make possible the development of amyloiddeposits in animals in a shorter period of time or to increase amyloiddeposits over a selected period of time. Amyloid-enhancing compounds maybe useful in screening assays for compounds which inhibit amyloidosis invivo, for example, in animal models, cellular assays and in vitro assaysfor amyloidosis. Such compounds may be used, for example, to providefaster or more sensitive assays for compounds. Modulation of amyloiddeposition is determined relative to an untreated subject or relative tothe treated subject prior to treatment.

“Inhibition” of amyloid deposition includes preventing or stopping ofamyloid formation, e.g., fibrillogenesis, clearance of amyloid, e.g.,soluble Aβ from brain, inhibiting or slowing down of further amyloiddeposition in a subject with amyloidosis, e.g., already having amyloiddeposits, and reducing or reversing amyloid fibrillogenesis or depositsin a subject with ongoing amyloidosis. Inhibition of amyloid depositionis determined relative to an untreated subject, or relative to thetreated subject prior to treatment, or, e.g., determined by clinicallymeasurable improvement, e.g., or in the case of a subject with brainamyloidosis, e.g., an Alzheimer's or cerebral amyloid angiopathysubject, stabilization of cognitive function or prevention of a furtherdecrease in cognitive function (i.e., preventing, slowing, or stoppingdisease progression), or improvement of parameters such as theconcentration of Aβ or tau in the CSF.

As used herein, “treatment” of a subject includes the application oradministration of a composition of the invention to a subject, orapplication or administration of a composition of the invention to acell or tissue from a subject, who has an amyloid-related disease orcondition, has a symptom of such a disease or condition, or is at riskof (or susceptible to) such a disease or condition, with the purpose ofcuring, healing, alleviating, relieving, altering, remedying,ameliorating, improving, or affecting the disease or condition, thesymptom of the disease or condition, or the risk of (or susceptibilityto) the disease or condition. The term “treating” refers to any indiciaof success in the treatment or amelioration of an injury, pathology orcondition, including any objective or subjective parameter such asabatement; remission; diminishing of symptoms or making the injury,pathology or condition more tolerable to the subject; slowing in therate of degeneration or decline; making the final point of degenerationless debilitating; improving a subject's physical or mental well-being;or, in some situations, preventing the onset of dementia. The treatmentor amelioration of symptoms can be based on objective or subjectiveparameters; including the results of a physical examination, apsychiatric evaluation, or a cognition test such as CDR, MMSE, DAD,ADAS-Cog, or another test known in the art. For example, the methods ofthe invention successfully treat a subject's dementia by slowing therate of or lessening the extent of cognitive decline.

In one embodiment, the term “treating” includes maintaining a subject'sCDR rating at its base line rating or at 0. In another embodiment, theterm treating includes decreasing a subject's CDR rating by about 0.25or more, about 0.5 or more, about 1.0 or more, about 1.5 or more, about2.0 or more, about 2.5 or more, or about 3.0 or more. In anotherembodiment, the term “treating” also includes reducing the rate of theincrease of a subject's CDR rating as compared to historical controls.In another embodiment, the term includes reducing the rate of increaseof a subject's CDR rating by about 5% or more, about 10% or more, about20% or more, about 25% or more, about 30% or more, about 40% or more,about 50% or more, about 60% or more, about 70% or more, about 80% ormore, about 90% or more, or about 100%, of the increase of thehistorical or untreated controls.

In another embodiment, the term “treating” also includes maintaining asubject's score on the MMSE. The term “treating” includes increasing asubject's MMSE score by about 1, about 2, about 3, about 4, about 5,about 7.5, about 10, about 12.5, about 15, about 17.5, about 20, orabout 25 points. The term also includes reducing the rate of thedecrease of a subject's MMSE score as compared to historical controls.In another embodiment, the term includes reducing the rate of decreaseof a subject's MMSE score by about 5% or less, about 10% or less, about20% or less, about 25% or less, about 30% or less, about 40% or less,about 50% or less, about 60% or less, about 70% or less, about 80% orless, about 90% or less or about 100% or less, of the decrease of thehistorical or untreated controls.

In another embodiment, the term “treating” also includes maintaining asubject's score on the DAD. The term “treating” includes increasing asubject's DAD score by about 1, about 5, about 10, about 15, about 20,about 30, about 35, about 40, about 50, about 60, about 70, or about 80points. The term also includes reducing the rate of the decrease of asubject's DAD score as compared to historical controls. In anotherembodiment, the term includes reducing the rate of decrease of asubject's DAD score by about 5% or less, about 10% or less, about 20% orless, about 25% or less, about 30% or less, about 40% or less, about 50%or less, about 60% or less, about 70% or less, about 80% or less, about90% or less or about 100% or less, of the decrease of the historical oruntreated controls.

In yet another embodiment, the term “treating” includes maintaining asubject's score on the ADAS-Cog. The term “treating” includes decreasinga subject's ADAS-Cog score by about 1 point or greater, by about 2points or greater, by about 3 points or greater, by about 4 points orgreater, by about 5 points or greater, by about 7.5 points or greater,by about 10 points or greater, by about 12.5 points or greater, by about15 points or greater, by about 17.5 points or greater, by about 20points or greater, or by about 25 points or greater. The term alsoincludes reducing the rate of the increase of a subject's ADAS-Cog scoreas compared to historical controls. In another embodiment, the termincludes reducing the rate of increase of a subject's ADAS-Cog score byabout 5% or more, about 10% or more, about 20% or more, about 25% ormore, about 30% or more, about 40% or more, about 50% or more, about 60%or more, about 70% or more, about 80% or more, about 90% or more orabout 100% of the increase of the historical or untreated controls.

In another embodiment, the term “treating” e.g., for AA or ALamyloidosis, includes an increase in serum creatinine, e.g., an increaseof creatinine clearance of 10% or greater, 20% or greater, 50% orgreater, 80% or greater, 90% or greater, 100% or greater, 150% orgreater, 200% or greater. The term “treating” also may induce remissionof nephrotic syndrome (NS). It may also include remission of chronicdiarrhea and/or a gain in body weight, e.g., by 10% or greater, 15% orgreater, or 20% or greater.

Without wishing to be bound by theory, in some aspects thepharmaceutical compositions of the invention contain a compound thatprevents or inhibits amyloid fibril formation, either in the brain orother organ of interest (acting locally) or throughout the entire body(acting systemically). Pharmaceutical compositions of the invention maybe effective in controlling amyloid deposition either following theirentry into the brain (following penetration of the blood brain bather)or from the periphery. When acting from the periphery, a compound of apharmaceutical composition may alter the equilibrium of amyloidogenicpeptide between the brain and the plasma so as to favor the exit ofamyloidogenic peptide from the brain. It may also favor clearance (orcatabolism) of the amyloid protein (soluble), and then prevent amyloidfibril formation and deposition due to a reduction of the amyloidprotein pool in a specific organ, e.g., liver, spleen, pancreas, kidney,joints, brain, etc. An increase in the exit of amyloidogenic peptidefrom the brain would result in a decrease in amyloidogenic peptide brainconcentration and therefore favor a decrease in amyloidogenic peptidedeposition. In particular, an agent may lower the levels of amyloid βpeptides, e.g., both Aβ40 and Aβ42 in the CSF and the plasma, or theagent may lower the levels of amyloid β peptides, e.g., Aβ 40 and Aβ42in the CSF and increase it in the plasma. Alternatively, compounds thatpenetrate the brain could control deposition by acting directly on brainamyloidogenic peptide e.g., by maintaining it in a non-fibrillar form orfavoring its clearance from the brain, by increasing its degradation inthe brain, or protecting brain cells from the detrimental effect ofamyloidogenic peptide. An agent can also cause a decrease of theconcentration of the amyloid protein (i.e., in a specific organ so thatthe critical concentration needed to trigger amyloid fibril formation ordeposition is not reached). Furthermore, the compounds described hereinmay inhibit or reduce an interaction between amyloid and a cell surfaceconstituent, for example, a glycosaminoglycan or proteoglycanconstituent of a basement membrane, whereby inhibiting or reducing thisinteraction produces the observed neuroprotective and cell-protectiveeffects. For example, the compound may also prevent an amyloid peptidefrom binding or adhering to a cell surface, a process which is known tocause cell damage or toxicity. Similarly, the compound may blockamyloid-induced cellular toxicity or microglial activation oramyloid-induced neurotoxicity, or inhibit amyloid induced inflammation.The compound may also reduce the rate or amount of amyloid aggregation,fibril formation, or deposition, or the compound lessens the degree ofamyloid deposition. The foregoing mechanisms of action should not beconstrued as limiting the scope of the invention inasmuch as theinvention may be practiced without such information.

The Aβ peptide has been shown by several groups to be highly toxic toneurons. Amyloid plaques are directly associated with reactive gliosis,dystrophic neurites and apoptotic cells, suggesting that plaques induceneurodegenerative changes. Neurotoxicity may eventually disrupt or evenkill neurons. In vitro, Aβ has been shown to induce apoptosis in manydifferent neuronal cell types, such as rat PC-12 cells, primary rathippocampal and cortical cultures, and the predifferentiated humanneurotype SH-SY5Y cell line (Dickson D W (2004) J Clin Invest 114:23-7;Canu et al. (2003) Cerebellum 2:270-278; Li et al. (1996) Brain Research738:196-204). Numerous reports have shown that Aβ fibrils can induceneurodegeneration, and it has been shown that neuronal cells exposed invitro to Aβ can become apoptotic (Morgan et al. (2004) Prog. Neurobiol.74:323-349; Stefani et al. (2003) J. Mol. Med. 81:678-99; La Ferla etal. (1997) J. Clin. Invest. 100(2):310-320). In Alzheimer's disease, aprogressive neuronal cell loss accompanies the deposition of Aβ amyloidfibrils in senile plaques.

In yet another aspect, the invention pertains to a method for inhibitingAβ-induced neuronal cell death by administering an effective amount of acompound of the present invention.

Another aspect of the invention pertains to a method for providingneuroprotection to a subject having an Aβ-amyloid related disease, e.g.,Alzheimer's disease, that includes administering an effective amount ofa compound of the present invention to the subject, such thatneuroprotection is provided.

In another aspect, methods for inhibiting Aβ-induced neuronal cell deathare provided that include administration of an effective amount of acompound of the present invention to a subject such that neuronal celldeath is inhibited.

In another aspect, methods for treating a disease state characterized byAβ-induced neuronal cell death in a subject are provided, e.g., byadministering an effective amount of a compound of the presentinvention. Non-limiting examples of such disease states includeAlzheimer's disease and Aβ-amyloid related diseases.

The term “neuroprotection” includes protection of neuronal cells of asubject from Aβ-induced cell death, e.g., cell death induced directly orindirectly by an Aβ peptide. Aβ-induced cell death may result ininitiation of processes such as, for example: the destabilization of thecytoskeleton; DNA fragmentation; the activation of hydrolytic enzymes,such as phospholipase A2; activation of caspases, calcium-activatedproteases and/or calcium-activated endonucleases; inflammation mediatedby macrophages; calcium influx into a cell; membrane potential changesin a cell; the disruption of cell junctions leading to decreased orabsent cell-cell communication; and the activation of expression ofgenes involved in cell death, e.g., immediate-early genes.

The term “amyloid-β disease” (or “amyloidβ related disease,” which termsas used herein are synonymous) may be used for mild cognitiveimpairment; vascular dementia; early Alzheimer's disease; Alzheimer'sdisease, including sporadic (non-hereditary) Alzheimer's disease andfamilial (hereditary) Alzheimer's disease; age-related cognitivedecline; cerebral amyloid angiopathy (“CAA”); hereditary cerebralhemorrhage; senile dementia; Down's syndrome; inclusion body myositis(“IBM”); or age-related macular degeneration (“ARMD”). According tocertain aspects of the invention, amyloid-β is a peptide having 39-43amino-acids, or amyloid-β is an amyloidogenic peptide produced fromβAPP.

Mild cognitive impairment (“MCI”) is a condition characterized by astate of mild but measurable impairment in thinking skills, which is notnecessarily associated with the presence of dementia. MCI frequently,but not necessarily, precedes Alzheimer's disease. It is a diagnosisthat has most often been associated with mild memory problems, but itcan also be characterized by mild impairments in other thinking skills,such as language or planning skills. However, in general, an individualwith MCI will have more significant memory lapses than would be expectedfor someone of their age or educational background. As the conditionprogresses, a physician may change the diagnosis to “Mild-to-ModerateCognitive Impairment,” as is well understood in this art.

Cerebral amyloid angiopathy (“CAA”) refers to the specific deposition ofamyloid fibrils in the walls of leptomingeal and cortical arteries,arterioles and in capillaries and veins. It is commonly associated withAlzheimer's disease, Down's syndrome and normal aging, as well as with avariety of familial conditions related to stroke or dementia (seeFrangione, et al., Amyloid: J. Protein Folding Disord. 8, Suppl. 1,36-42 (2001)). CAA can occur sporadically or be hereditary. Multiplemutation sites in either Aβ or the APP gene have been identified and areclinically associated with either dementia or cerebral hemorrhage.Exemplary CAA disorders include, but are not limited to, hereditarycerebral hemorrhage with amyloidosis of Icelandic type (HCHWA-I); theDutch variant of HCHWA (HCHWA-D; a mutation in Aβ); the Flemish mutationof Aβ; the Arctic mutation of Aβ; the Italian mutation of Aβ; the Iowamutation of Aβ; familial British dementia; and familial Danish dementia.Cerebral amyloid angiopathy is known to be associated with cerebralhemorrhage (or hemorrhagic stroke).

Additionally, abnormal accumulation of APP and of amyloid-β protein inmuscle fibers has been implicated in the pathology of sporadic inclusionbody myositis (“IBM”) (Askanas, et al., Proc. Natl. Acad. Sci. USA 93,1314-19 (1996); Askanas, et al., Current Opinion in Rheumatology 7,486-96 (1995)). Accordingly, the compounds of the invention can be usedprophylactically or therapeutically in the treatment of disorders inwhich amyloid-β protein is abnormally deposited at non-neurologicallocations, such as treatment of IBM by delivery of the compounds tomuscle fibers.

Additionally, it has been shown that Aβ is associated with abnormalextracellular deposits, known as drusen, that accumulate along the basalsurface of the retinal pigmented epithelium in individuals withage-related macular degeneration (ARMD). ARMD is a cause of irreversiblevision loss in older individuals. It is believed that Aβ depositioncould be an important component of the local inflammatory events thatcontribute to atrophy of the retinal pigmented epithelium, drusenbiogenesis, and the pathogenesis of ARMD (Johnson, et al., Proc. Natl.Acad. Sci. USA 99(18), 11830-5 (2002)). Therefore, the invention alsorelates to the treatment or prevention of age-related maculardegeneration.

Also, the invention relates to a method for preventing or inhibitingamyloid deposition in a subject. For example, such a method comprisesadministering to a subject a therapeutically effective amount of acompound capable of reducing the concentration of amyloid (e.g., ALamyloid protein (λ or κ-chain related, e.g., amyloid λ, amyloid κ,amyloid κIV, amyloid λVI, amyloid γ, amyloid γ1), Aβ, IAPP, β₂m, AA, AHamyloid protein, or other amyloids), such that amyloid accumulation ordeposition is prevented or inhibited.

In another aspect, the invention relates to a method for preventing,reducing, or inhibiting amyloid deposition in a subject. For example,such a method comprises administering to a subject a therapeuticallyeffective amount of a compound capable of inhibiting amyloid (e.g., ALamyloid protein (λ or κ-chain related, e.g., amyloid λ, amyloid κ,amyloid κIV, amyloid λVI, amyloid γ, amyloid γ1), Aβ, IAPP, β₂m, AA, AHamyloid protein, or other amyloids), such that amyloid deposition isprevented, reduced, or inhibited.

The invention also relates to a method for modulating, e.g., minimizing,amyloid-associated damage to cells, comprising the step of administeringa compound capable of reducing the concentration of amyloid (e.g., ALamyloid protein (λ or κ-chain related, e.g., amyloid λ, amyloid κ,amyloid κIV, amyloid λVI, amyloid γ, amyloid γ1), Aβ, IAPP, β₂m, AA, AHamyloid protein, or another amyloid), such that said amyloid-associateddamage to cells is modulated. In certain aspects of the invention, themethods for modulating amyloid-associated damage to cells comprise astep of administering a compound capable of reducing the concentrationof amyloid or reducing the interaction of an amyloid with a cellsurface.

The invention also includes a method for directly or indirectlypreventing cell death in a subject, the method comprising administeringto a subject a therapeutically effective amount of a compound capable ofpreventing amyloid (e.g., AL amyloid protein (λ or κ-chain related,e.g., amyloid λ, amyloid κ, amyloid κIV, amyloid λVI, amyloid γ, amyloidγ1), Aβ, IAPP, β₂m, AA, AH amyloid protein, or other amyloid) mediatedevents that lead, directly or indirectly, to cell death.

In an embodiment, the method is used to treat Alzheimer's disease (e.g.sporadic or familial AD). The method can also be used prophylacticallyor therapeutically to treat other clinical occurrences of amyloid-βdeposition, such as in Down's syndrome individuals and in patients withcerebral amyloid angiopathy (“CAA”) or hereditary cerebral hemorrhage.

The compounds of the invention may be used prophylactically ortherapeutically in the treatment of disorders in which amyloid-betapeptide is abnormally deposited at non-neurological locations, such astreatment of IBM by delivery of the compounds to muscle fibers, ortreatment of macular degeneration by delivery of the compound(s) of theinvention to the basal surface of the retinal pigmented epithelium.

The present invention also provides a method for modulatingamyloid-associated damage to cells, comprising the step of administeringa compound capable of reducing the concentration of Aβ, or capable ofminimizing the interaction of Aβ (soluble oligomeric or fibrillary) withthe cell surface, such that said amyloid-associated damage to cells ismodulated. In certain aspects of the invention, the methods formodulating amyloid-associated damage to cells comprise a step ofadministering a compound capable of reducing the concentration of Aβ orreducing the interaction of Aβ with a cell surface.

In accordance with the present invention, there is further provided amethod for preventing cell death in a subject, said method comprisingadministering to a subject a therapeutically effective amount of acompound capable of preventing Aβ-mediated events that lead, directly orindirectly, to cell death.

The present invention also provides a method for modulatingamyloid-associated damage to cells, comprising the step of administeringa compound capable of reducing the concentration of IAPP, or capable ofminimizing the interaction of IAPP (soluble oligomeric or fibrillary)with the cell surface, such that said amyloid-associated damage to cellsis modulated. In certain aspects of the invention, the methods formodulating amyloid-associated damage to cells comprise a step ofadministering a compound capable of reducing the concentration of IAPPor reducing the interaction of IAPP with a cell surface.

In accordance with the present invention, there is further provided amethod for preventing cell death in a subject, said method comprisingadministering to a subject a therapeutically effective amount of acompound capable of preventing IAPP-mediated events that lead, directlyor indirectly, to cell death.

This invention also provides methods and compositions which are usefulin the treatment of amyloidosis. The methods of the invention involveadministering to a subject a therapeutic compound which inhibits amyloiddeposition. Accordingly, the compositions and methods of the inventionare useful for inhibiting amyloidosis in disorders in which amyloiddeposition occurs. The methods of the invention can be usedtherapeutically to treat amyloidosis or can be used prophylactically ina subject susceptible to (hereditary) amyloidosis or identified as beingat risk to develop amyloidosis, e.g., hereditary, or identified as beingat risk to develop amyloidosis. In certain embodiments, the inventionincludes a method of inhibiting an interaction between an amyloidogenicprotein and a constituent of basement membrane to inhibit amyloiddeposition. The constituent of basement membrane is a glycoprotein orproteoglycan, preferably heparan sulfate proteoglycan. A therapeuticcompound used in this method may interfere with binding of a basementmembrane constituent to a target binding site on an amyloidogenicprotein, thereby inhibiting amyloid deposition.

In some aspects, the methods of the invention involve administering to asubject a therapeutic compound which inhibits amyloid deposition“Inhibition of amyloid deposition,” includes the prevention of amyloidformation, inhibition of further amyloid deposition in a subject withongoing amyloidosis and reduction of amyloid deposits in a subject withongoing amyloidosis Inhibition of amyloid deposition is determinedrelative to an untreated subject or relative to the treated subjectprior to treatment. In an embodiment, amyloid deposition is inhibited byinhibiting an interaction between an amyloidogenic protein and aconstituent of basement membrane. “Basement membrane” refers to anextracellular matrix comprising glycoproteins and proteoglycans,including laminin, collagen type IV, fibronectin, perlecan, agrin,dermatan sulfate, and heparan sulfate proteoglycan (HSPG). In oneembodiment, amyloid deposition is inhibited by interfering with aninteraction between an amyloidogenic protein and a sulfatedglycosaminoglycan such as HSPG, dermatan sulfate, perlecan or agrinsulfate. Sulfated glycosaminoglycans are known to be present in alltypes of amyloids (see Snow, et al. Lab. Invest. 56, 120-23 (1987)) andamyloid deposition and HSPG deposition occur coincidentally in animalmodels of amyloidosis (see Snow, et al. Lab. Invest. 56, 665-75 (1987)and Gervais, F. et al. Curr. Med. Chem., 3, 361-370 (2003)). Consensusbinding site motifs for HSPG in amyloidogenic proteins have beendescribed (see, e.g., Cardin and Weintraub Arteriosclerosis 9, 21-32(1989)).

The ability of a compound to prevent or block the formation ordeposition of amyloid may reside in its ability to bind tonon-fibrillar, soluble amyloid protein and to maintain its solubility.

The ability of a therapeutic compound of the invention to inhibit aninteraction between an amyloidogenic protein and a glycoprotein orproteoglycan constituent of a basement membrane can be assessed by an invitro binding assay, such as that described in U.S. Pat. No. 5,164,295,the contents of which are hereby incorporated by reference.Alternatively, the ability of a compound to bind to an amyloidogenicprotein or to inhibit the binding of a basement membrane constituent(e.g., HSPG) to an amyloidogenic protein (e.g., Aβ) can be measuredusing a mass spectrometry assay where soluble protein, e.g., Aβ, IAPP,β₂M is incubated with the compound. A compound which binds to, e.g., Aβ,will induce a change in the mass spectrum of the protein. Exemplaryprotocols for a mass spectrometry assay employing Aβ and IAPP can befound in the Examples, the results of which are provided in Table 3. Theprotocol can readily be modified to adjust the sensitivity of the data,e.g., by adjusting the amount of protein and/or compound employed. Thus,e.g., binding might be detected for test compounds noted as not havingdetectable binding employing less sensitive test protocols.

Alternative methods for screening compounds exist and can readily beemployed by a skilled practitioner to provide an indication of theability of test compounds to bind to, e.g., fibrillar Aβ. One suchscreening assay is an ultraviolet absorption assay. In an exemplaryprotocol, a test compound (20 μM) is incubated with 50 μM Aβ(1-40)fibers for 1 hour at 37° C. in Tris buffered saline (20 mM Tris, 150 mMNaCl, pH 7.4 containing 0.01 sodium azide). Following incubation, thesolution is centrifuged for 20 minutes at 21,000 g to sediment theAβ(1-40) fibers along with any bound test compound. The amount of testcompound remaining in the supernatant can then be determined by readingthe absorbance. The fraction of test compound bound can then becalculated by comparing the amount remaining in the supernatants ofincubations with Aβ to the amount remaining in control incubations whichdo not contain Aβ fibers. Thioflavin T and Congo Red, both of which areknown to bind to Aβ fibers, may be included in each assay run aspositive controls. Before assaying, test compounds can be diluted to 40μM, which would be twice the concentration in the final test, and thenscanned using the Hewlett Packard 8453 UV/VIS spectrophotometer todetermine if the absorbance is sufficient for detection.

In another embodiment, the invention pertains to a method for improvingcognition in a subject suffering from an amyloid-related disease. Themethod includes administering an effective amount of a therapeuticcompound of the invention, such that the subject's cognition isimproved. The subject's cognition can be tested using methods known inthe art such as the Clinical Dementia Rating (“CDR”), Mini-Mental StateExamination (“MMSE”), Disability Assessment for Dementia (“DAD”), and/orthe Alzheimer's Disease Assessment Scale-Cognition (“ADAS-Cog”).

In another embodiment, the invention pertains to a method for treating asubject for an amyloid-related disease. The method includesadministering a cognitive test to a subject prior to administration of acompound of the invention, administering an effective amount of acompound of the invention to the subject, and administering a cognitivetest to the subject subsequent to administration of the compound, suchthat the subject is treated for the amyloid-related disease, wherein thesubject's score on said cognitive test is improved.

“Improvement,” “improved” or “improving” in cognition is present withinthe context of the present invention if there is a statisticallysignificant difference in the direction of normality between theperformance of subjects treated using the methods of the invention ascompared to members of a placebo group, historical control, or betweensubsequent tests given to the same subject.

In one embodiment, a subject's CDR is maintained at 0. In anotherembodiment, a subject's CDR is decreased (e.g., improved) by about 0.25or more, about 0.5 or more, about 1.0 or more, about 1.5 or more, about2.0 or more, about 2.5 or more, or about 3.0 or more. In anotherembodiment, the rate of increase of a subject's CDR rating is reduced byabout 5% or more, about 10% or more, about 20% or more, about 25% ormore, about 30% or more, about 40% or more, about 50% or more, about 60%or more, about 70% or more, about 80% or more, about 90% or more, orabout 100% or more of the increase of the historical or untreatedcontrols.

In one embodiment, a subject's score on the MMSE is maintained.Alternatively, the subject's score on the MMSE may be increased by about1, about 2, about 3, about 4, about 5, about 7.5, about 10, about 12.5,about 15, about 17.5, about 20, or about 25 points. In anotheralternative, the rate of the decrease of a subject's MMSE score ascompared to historical controls is reduced. For example, the rate of thedecrease of a subject's MMSE score may be reduced by about 5% or more,about 10% or more, about 20% or more, about 25% or more, about 30% ormore, about 40% or more, about 50% or more, about 60% or more, about 70%or more, about 80% or more, about 90% or more, or about 100% or more ofthe decrease of the historical or untreated controls.

In one embodiment, a subject's score on the DAD is maintained.Alternatively, the subject's score on the DAD may be increased by about1, about 2, about 3, about 4, about 5, about 7.5, about 10, about 15,about 20, about 30, about 40, or about 50 or more points. In anotheralternative, the rate of the decrease of a subject's DAD score ascompared to historical controls is reduced. For example, the rate of thedecrease of a subject's DAD score may be reduced by about 5% or more,about 10% or more, about 20% or more, about 25% or more, about 30% ormore, about 40% or more, about 50% or more, about 60% or more, about 70%or more, about 80% or more, about 90% or more, or about 100% or more ofthe decrease of the historical or untreated controls.

In one embodiment, the invention pertains to a method for treating,slowing or stopping an amyloid-related disease associated with cognitiveimpairment, by administering to a subject an effective amount of atherapeutic compound of the invention, wherein the annual deteriorationof the subject's cognition as measured by ADAS-Cog is less than 8 pointsper year, less the 6 points per year, less than 5 points per year, lessthan 4 points per year, or less than 3 points per year. In a furtherembodiment, the invention pertains to a method for treating, slowing orstopping an amyloid-related disease associated with cognition byadministering an effective amount of a therapeutic compound of theinvention such that the subject's cognition as measured by ADAS-Cogremains constant over a year. “Constant” includes fluctuations of nomore than 2 points. Remaining constant includes fluctuations of twopoints or less in either direction. In a further embodiment, thesubject's cognition improves by 2 points or greater per year, 3 pointsor greater per year, 4 point or greater per year, 5 points or greaterper year, 6 points or greater per year, 7 points or greater per year, 8points or greater per year, etc. as measured by the ADAS-Cog. In anotheralternative, the rate of the increase of a subject's ADAS-Cog score ascompared to historical controls is reduced. For example, the rate of theincrease of a subject's ADAS-Cog score may be reduced by about 5% ormore, about 10% or more, about 20% or more, about 25% or more, about 30%or more, about 40% or more, about 50% or more, about 60% or more, about70% or more, about 80% or more, about 90% or more or about 100% of theincrease of the historical or untreated controls.

In another embodiment, the ratio of Aβ42:Aβ40 in the CSF or plasma of asubject decreases by about 15% or more, about 20% or more, about 25% ormore, about 30% or more, about 35% or more, about 40% or more, about 45%or more, or about 50% or more. In another embodiment, the levels of Aβin the subject's cerebrospinal fluid decrease by about 15% or more,about 25% or more, about 35% or more, about 45% or more, about 55% ormore, about 75% or more, or about 90% or more.

It is to be understood that wherever values and ranges are providedherein, e.g., in ages of subject populations, dosages, and blood levels,all values and ranges encompassed by these values and ranges, are meantto be encompassed within the scope of the present invention. Moreover,all values in these values and ranges may also be the upper or lowerlimits of a range.

Furthermore, the invention pertains to any novel chemical compounddescribed herein. That is, the invention relates to novel compounds, andnovel methods of their use as described herein, which are within thescope of the Formulae disclosed herein, and which are not disclosed inthe cited Patents and patent applications.

Synthesis of Compounds of the Invention

In general, the compounds of the present invention may be prepared bythe methods illustrated in the general reaction schemes as, for example,described below, or by modifications thereof, using readily availablestarting materials, reagents and conventional synthesis procedures. Inthese reactions, it is also possible to make use of variants which arein themselves known, but are not mentioned here. Functional andstructural equivalents of the compounds described herein and which havethe same general properties, wherein one or more simple variations ofsubstituents are made which do not adversely affect the essential natureor the utility of the compound are also included.

The compounds of the present invention may be readily prepared inaccordance with the synthesis schemes and protocols described herein, asillustrated in the specific procedures provided. However, those skilledin the art will recognize that other synthetic pathways for forming thecompounds of this invention may be used, and that the following isprovided merely by way of example, and is not limiting to the presentinvention. See, e.g., “Comprehensive Organic Transformations” by R.Larock, VCH Publishers (1989). It will be further recognized thatvarious protecting and deprotecting strategies will be employed that arestandard in the art (See, e.g., “Protective Groups in Organic Synthesis”by Greene and Wuts). Those skilled in the relevant arts will recognizethat the selection of any particular protecting group (e.g., amine andcarboxyl protecting groups) will depend on the stability of theprotected moiety with regards to the subsequent reaction conditions andwill understand the appropriate selections.

Further illustrating the knowledge of those skilled in the art is thefollowing sampling of the extensive chemical literature: “Chemistry ofthe Amino Acids” by J.P. Greenstein and M. Winitz, John Wiley & Sons,Inc., New York (1961); “Comprehensive Organic Transformations” by R.Larock, VCH Publishers (1989); T. D. Ocain, et al., J. Med. Chem. 31,2193-99 (1988); E. M. Gordon, et al., J. Med. Chem. 31, 2199-10 (1988);“Practice of Peptide Synthesis” by M. Bodansky and A. Bodanszky,Springer-Verlag, New York (1984); “Protective Groups in OrganicSynthesis” by T. Greene and P. Wuts (1991); “Asymmetric Synthesis:Construction of Chiral Molecules Using Amino Acids” by G. M. Coppola andH. F. Schuster, John Wiley & Sons, Inc., New York (1987); “The ChemicalSynthesis of Peptides” by J. Jones, Oxford University Press, New York(1991); and “Introduction of Peptide Chemistry” by P. D. Bailey, JohnWiley & Sons, Inc., New York (1992).

The synthesis of compounds of the invention is carried out in a solvent.Suitable solvents are liquids at ambient room temperature and pressureor remain in the liquid state under the temperature and pressureconditions used in the reaction. Useful solvents are not particularlyrestricted provided that they do not interfere with the reaction itself(that is, they preferably are inert solvents), and they dissolve acertain amount of the reactants. Depending on the circumstances,solvents may be distilled or degassed. Solvents may be, for example,aliphatic hydrocarbons (e.g., hexanes, heptanes, ligroin, petroleumether, cyclohexane, or methylcyclohexane) and halogenated hydrocarbons(e.g., methylenechloride, chloroform, carbontetrachloride,dichloroethane, chlorobenzene, or dichlororbenzene); aromatichydrocarbons (e.g., benzene, toluene, tetrahydronaphthalene,ethylbenzene, or xylene); ethers (e.g., diglyme, methyl-tert-butylether, methyl-tert-amyl ether, ethyl-tert-butyl ether, diethylether,diisopropylether, tetrahydrofuran or methyltetrahydrofurans, dioxane,dimethoxyethane, or diethyleneglycol dimethylether); nitriles (e.g.,acetonitrile); ketones (e.g., acetone); esters (e.g., methyl acetate orethyl acetate); and mixtures thereof.

In general, after completion of the reaction, the product is isolatedfrom the reaction mixture according to standard techniques. For example,the solvent is removed by evaporation or filtration if the product issolid, optionally under reduced pressure. After the completion of thereaction, water may be added to the residue to make the aqueous layeracidic or basic and the precipitated compound filtered, although careshould be exercised when handling water-sensitive compounds. Similarly,water may be added to the reaction mixture with a hydrophobic solvent toextract the target compound. The organic layer may be washed with water,dried over anhydrous magnesium sulphate or sodium sulphate, and thesolvent is evaporated to obtain the target compound. The target compoundthus obtained may be purified, if necessary, e.g., by recrystallization,reprecipitation, chromatography, or by converting it to a salt byaddition of an acid or base.

The compounds of the invention may be supplied in a solution with anappropriate solvent or in a solvent-free form (e.g., lyophilized). Inanother aspect of the invention, the compounds and buffers necessary forcarrying out the methods of the invention may be packaged as a kit,optionally including a container. The kit may be commercially used fortreating or preventing amyloid-related disease according to the methodsdescribed herein and may include instructions for use in a method of theinvention. Additional kit components may include acids, bases, bufferingagents, inorganic salts, solvents, antioxidants, preservatives, or metalchelators. The additional kit components are present as purecompositions, or as aqueous or organic solutions that incorporate one ormore additional kit components. Any or all of the kit componentsoptionally further comprise buffers.

The term “container” includes any receptacle for holding the therapeuticcompound. For example, in one embodiment, the container is the packagingthat contains the compound. In other embodiments, the container is notthe packaging that contains the compound, i.e., the container is areceptacle, such as a box or vial that contains the packaged compound orunpackaged compound and the instructions for use of the compound.Moreover, packaging techniques are well known in the art. It should beunderstood that the instructions for use of the therapeutic compound maybe contained on the packaging containing the therapeutic compound, andas such the instructions form an increased functional relationship tothe packaged product.

Pharmaceutical Preparations

In another embodiment, the present invention relates to pharmaceuticalcompositions comprising agents according to any of the Formulae hereinfor the treatment of an amyloid-related disease, as well as methods ofmanufacturing such pharmaceutical compositions.

In general, the agents of the present invention may be prepared by themethods illustrated in the general reaction schemes as, for example, inthe patents and patent applications referred to herein, or bymodifications thereof, using readily available starting materials,reagents and conventional synthesis procedures. In these reactions, itis also possible to make use of variants which are in themselves known,but are not mentioned here. Functional and structural equivalents of theagents described herein and which have the same general properties,wherein one or more simple variations of substituents are made which donot adversely affect the essential nature or the utility of the agentare also included.

The agents of the invention may be supplied in a solution with anappropriate solvent or in a solvent-free form (e.g., lyophilized). Inanother aspect of the invention, the agents and buffers necessary forcarrying out the methods of the invention may be packaged as a kit. Thekit may be commercially used according to the methods described hereinand may include instructions for use in a method of the invention.Additional kit components may include acids, bases, buffering agents,inorganic salts, solvents, antioxidants, preservatives, or metalchelators. The additional kit components are present as purecompositions, or as aqueous or organic solutions that incorporate one ormore additional kit components. Any or all of the kit componentsoptionally further comprise buffers.

The therapeutic agent may also be administered parenterally,intraperitoneally, intraspinally, or intracerebrally. Dispersions can beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations may contain a preservative to prevent the growth ofmicroorganisms.

To administer the therapeutic agent by other than parenteraladministration, it may be necessary to coat the agent with, orco-administer the agent with, a material to prevent its inactivation.For example, the therapeutic agent may be administered to a subject inan appropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Liposomes include water-in-oil-in-water CGF emulsions as wellas conventional liposomes (Strejan et al., J. Neuroimmunol. 7, 27(1984)).

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases, the composition must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi.

Suitable pharmaceutically acceptable vehicles include, withoutlimitation, any non-immunogenic pharmaceutical adjuvants suitable fororal, parenteral, nasal, mucosal, transdermal, intravascular (IV),intraarterial (IA), intramuscular (IM), and subcutaneous (SC)administration routes, such as phosphate buffer saline (PBS).

The vehicle can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, isotonic agents are included, for example, sugars, sodiumchloride, or polyalcohols such as mannitol and sorbitol, in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate or gelatin.

Sterile injectable solutions can be prepared by incorporating thetherapeutic agent in the required amount in an appropriate solvent withone or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the therapeutic agent into a sterile vehicle whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the methods of preparationare vacuum drying and freeze-drying which yields a powder of the activeingredient (i.e., the therapeutic agent) plus any additional desiredingredient from a previously sterile-filtered solution thereof.

The therapeutic agent can be orally administered, for example, with aninert diluent or an assimilable edible carrier. The therapeutic agentand other ingredients may also be enclosed in a hard or soft shellgelatin capsule, compressed into tablets, or incorporated directly intothe subject's diet. For oral therapeutic administration, the therapeuticagent may be incorporated with excipients and used in the form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. The percentage of thetherapeutic agent in the compositions and preparations may, of course,be varied. The amount of the therapeutic agent in such therapeuticallyuseful compositions is such that a suitable dosage will be obtained.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontaining a predetermined quantity of therapeutic agent calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical vehicle. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the therapeutic agent and the particular therapeuticeffect to be achieved, and (b) the limitations inherent in the art ofcompounding such a therapeutic agent for the treatment of amyloiddeposition in subjects.

The present invention therefore includes pharmaceutical formulationscomprising the agents of the Formulae described herein, includingpharmaceutically acceptable salts thereof, in pharmaceuticallyacceptable vehicles for aerosol, oral and parenteral administration.Also, the present invention includes such agents, or salts thereof,which have been lyophilized and which may be reconstituted to formpharmaceutically acceptable formulations for administration, as byintravenous, intramuscular, or subcutaneous injection. Administrationmay also be intradermal or transdermal.

In accordance with the present invention, an agent of the Formulaedescribed herein, and pharmaceutically acceptable salts thereof, may beadministered orally or through inhalation as a solid, or may beadministered intramuscularly or intravenously as a solution, suspensionor emulsion. Alternatively, the agents or salts may also be administeredby inhalation, intravenously or intramuscularly as a liposomalsuspension.

Pharmaceutical formulations are also provided which are suitable foradministration as an aerosol, by inhalation. These formulations comprisea solution or suspension of the desired agent of any Formula herein, ora salt thereof, or a plurality of solid particles of the agent or salt.The desired formulation may be placed in a small chamber and nebulized.Nebulization may be accomplished by compressed air or by ultrasonicenergy to form a plurality of liquid droplets or solid particlescomprising the agents or salts. The liquid droplets or solid particlesshould have a particle size in the range of about 0.5 to about 5microns. The solid particles can be obtained by processing the solidagent of any Formula described herein, or a salt thereof, in anyappropriate manner known in the art, such as by micronization. The sizeof the solid particles or droplets will be, for example, from about 1 toabout 2 microns. In this respect, commercial nebulizers are available toachieve this purpose.

A pharmaceutical formulation suitable for administration as an aerosolmay be in the form of a liquid, the formulation will comprise awater-soluble agent of any Formula described herein, or a salt thereof,in a carrier which comprises water. A surfactant may be present whichlowers the surface tension of the formulation sufficiently to result inthe formation of droplets within the desired size range when subjectedto nebulization.

Peroral compositions also include liquid solutions, emulsions,suspensions, and the like. The pharmaceutically acceptable vehiclessuitable for preparation of such compositions are well known in the art.Typical components of carriers for syrups, elixirs, emulsions andsuspensions include ethanol, glycerol, propylene glycol, polyethyleneglycol, liquid sucrose, sorbitol and water. For a suspension, typicalsuspending agents include methyl cellulose, sodium carboxymethylcellulose, tragacanth, and sodium alginate; typical wetting agentsinclude lecithin and polysorbate 80; and typical preservatives includemethyl paraben and sodium benzoate. Peroral liquid compositions may alsocontain one or more components such as sweeteners, flavoring agents andcolorants disclosed above.

Pharmaceutical compositions may also be coated by conventional methods,typically with pH or time-dependent coatings, such that the subjectagent is released in the gastrointestinal tract in the vicinity of thedesired topical application, or at various times to extend the desiredaction. Such dosage forms typically include, but are not limited to, oneor more of cellulose acetate phthalate, polyvinylacetate phthalate,hydroxypropyl methyl cellulose phthalate, ethyl cellulose, waxes, andshellac.

Other compositions useful for attaining systemic delivery of the subjectagents include sublingual, buccal and nasal dosage forms. Suchcompositions typically comprise one or more of soluble filler substancessuch as sucrose, sorbitol and mannitol; and binders such as acacia,microcrystalline cellulose, carboxymethyl cellulose and hydroxypropylmethyl cellulose. Glidants, lubricants, sweeteners, colorants,antioxidants and flavoring agents disclosed above may also be included.

The compositions of this invention can also be administered topically toa subject, e.g., by the direct laying on or spreading of the compositionon the epidermal or epithelial tissue of the subject, or transdermallyvia a “patch”. Such compositions include, for example, lotions, creams,solutions, gels and solids. These topical compositions may comprise aneffective amount, usually at least about 0.1%, or even from about 1% toabout 5%, of an agent of the invention. Suitable carriers for topicaladministration typically remain in place on the skin as a continuousfilm, and resist being removed by perspiration or immersion in water.Generally, the carrier is organic in nature and capable of havingdispersed or dissolved therein the therapeutic agent. The carrier mayinclude pharmaceutically acceptable emollients, emulsifiers, thickeningagents, solvents and the like.

In one embodiment, active agents are administered at a therapeuticallyeffective dosage sufficient to inhibit amyloid deposition in a subject.A “therapeutically effective” dosage inhibits amyloid deposition by, forexample, at least about 20%, or by at least about 40%, or even by atleast about 60%, or by at least about 80% relative to untreatedsubjects. In the case of an Alzheimer's subject, a “therapeuticallyeffective” dosage stabilizes cognitive function or prevents a furtherdecrease in cognitive function (i.e., preventing, slowing, or stoppingdisease progression). The present invention accordingly providestherapeutic drugs. By “therapeutic” or “drug” is meant an agent having abeneficial ameliorative or prophylactic effect on a specific disease orcondition in a living human or non-human animal.

In the case of AA or AL amyloidosis, the agent may improve or stabilizespecific organ function. As an example, renal function may be stabilizedor improved by 10% or greater, 20% or greater, 30% or greater, 40% orgreater, 50% or greater, 60% or greater, 70% or greater, 80% or greater,or by greater than 90%.

In the case of IAPP, the agent may maintain or increase β-islet cellfunction, as determined by insulin concentration or the Pro-IAPP/IAPPratio. In a further embodiment, the Pro-IAPP/IAPP ratio is increased byabout 10% or greater, about 20% or greater, about 30% or greater, about40% or greater, or by about 50%. In a further embodiment, the ratio isincreased up to 50%. In addition, a therapeutically effective amount ofthe agent may be effective to improve glycemia or insulin levels.

In another embodiment, the active agents are administered at atherapeutically effective dosage sufficient to treat AA (secondary)amyloidosis and/or AL (primary) amyloidosis, by stabilizing renalfunction, decreasing proteinuria, increasing creatinine clearance (e.g.,by at least 50% or greater or by at least 100% or greater), remission ofchronic diarrhea, or by weight gain (e.g., 10% or greater). In addition,the agents may be administered at a therapeutically effective dosagesufficient to improve nephrotic syndrome.

Furthermore, active agents may be administered at a therapeuticallyeffective dosage sufficient to decrease deposition in a subject ofamyloid protein, e.g., Aβ40 or Aβ42. A therapeutically effective dosagedecreases amyloid deposition by, for example, at least about 15%, or byat least about 40%, or even by at least 60%, or at least by about 80%relative to untreated subjects.

In another embodiment, active agents are administered at atherapeutically effective dosage sufficient to increase or enhanceamyloid protein, e.g., Aβ40 or Aβ 42, in the blood, CSF, or plasma of asubject. A therapeutically effective dosage increases the concentrationby, for example, at least about 15%, or by at least about 40%, or evenby at least 60%, or at least by about 80% relative to untreatedsubjects.

In yet another embodiment, active agents are administered at atherapeutically effective dosage sufficient to maintain a subject's CDRrating at its base line rating or at 0. In another embodiment, theactive agents are administered at a therapeutically effective dosagesufficient to decrease a subject's CDR rating by about 0.25 or more,about 0.5 or more, about 1.0 or more, about 1.5 or more, about 2.0 ormore, about 2.5 or more, or about 3.0 or more. In another embodiment,the active agents are administered at a therapeutically effective dosagesufficient to reduce the rate of the increase of a subject's CDR ratingas compared to historical or untreated controls. In another embodiment,the therapeutically effective dosage is sufficient to reduce the rate ofincrease of a subject's CDR rating (relative to untreated subjects) byabout 5% or greater, about 10% or greater, about 20% or greater, about25% or greater, about 30% or greater, about 40% or greater, about 50% orgreater, about 60% or greater, about 70% or greater, about 80% orgreater, about 90% or greater or about 100% or greater.

In yet another embodiment, active agents are administered at atherapeutically effective dosage sufficient to maintain a subject'sscore on the MMSE. In another embodiment, the active agents areadministered at a therapeutically effective dosage sufficient toincrease a subject's MMSE score by about 1, about 2, about 3, about 4,about 5, about 7.5, about 10, about 12.5, about 15, about 17.5, about20, or about 25 points. In another embodiment, the active agents areadministered at a therapeutically effective dosage sufficient to reducethe rate of the decrease of a subject's MMSE score as compared tohistorical controls. In another embodiment, the therapeuticallyeffective dosage is sufficient to reduce the rate of decrease of asubject's MMSE score may be about 5% or less, about 10% or less, about20% or less, about 25% or less, about 30% or less, about 40% or less,about 50% or less, about 60% or less, about 70% or less, about 80% orless, about 90% or less or about 100% or less, of the decrease of thehistorical or untreated controls.

Another means to evaluate cognition, particularly Alzheimer's disease,is the Alzheimer's Disease Assessment Scale (ADAS-Cog), or a variationtermed the Standardized Alzheimer's Disease Assessment Scale (SADAS). Itis commonly used as an efficacy measure in clinical drug trials ofAlzheimer's disease and related disorders characterized by cognitivedecline. SADAS and ADAS-Cog were not designed to diagnose Alzheimer'sdisease; they are useful in characterizing symptoms of dementia and area relatively sensitive indicator of dementia progression. (See, e.g.,Doraiswamy, Neurology 48:1511-1517, 1997; and Standish, J. Am. Geriatr.Soc. 44:712-716, 1996.) Annual deterioration in untreated Alzheimer'sdisease patients is approximately 8 points per year (See, e.g., Raskind,M. Prim. Care Companion J Clin Psychiatry 2000 August; 2(4):134-138).

In yet another embodiment, active agents are administered at atherapeutically effective dosage sufficient to maintain a subject'sscore on the DAD. In another embodiment, the active agents areadministered at a therapeutically effective dosage sufficient toincrease a subject's DAD score by about 1, about 2, about 3, about 4,about 5, about 10, about 15, about 20, about 25, about 30, about 40, orabout 50 points or greater. In another embodiment, the active agents areadministered at a therapeutically effective dosage sufficient to reducethe rate of the decrease of a subject's DAD score as compared tohistorical controls. In another embodiment, the therapeuticallyeffective dosage is sufficient to reduce the rate of decrease of asubject's DAD score by about 5% or less, about 10% or less, about 20% orless, about 25% or less, about 30% or less, about 40% or less, about 50%or less, about 60% or less, about 70% or less, about 80% or less, about90% or less or about 100% or less, of the decrease of the historical oruntreated controls.

In yet another embodiment, active agents are administered at atherapeutically effective dosage sufficient to maintain a subject'sscore on the ADAS-Cog. In another embodiment, the active agents areadministered at a therapeutically effective dosage sufficient todecrease a subject's ADAS-Cog score by about 2 points or greater, byabout 3 points or greater, by about 4 points or greater, by about 5points or greater, by about 7.5 points or greater, by about 10 points orgreater, by about 12.5 points or greater, by about 15 points or greater,by about 17.5 points or greater, by about 20 points or greater, or byabout 25 points or greater. In another embodiment, the active agents areadministered at a therapeutically effective dosage sufficient to reducethe rate of the increase of a subject's ADAS-Cog scores as compared tohistorical or untreated controls. In another embodiment, thetherapeutically effective dosage is sufficient to reduce the rate ofincrease of a subject's ADAS-Cog scores (relative to untreated subjects)by about 5% or greater, about 10% or greater, about 20% or greater,about 25% or greater, about 30% or greater, about 40% or greater, about50% or greater, about 60% or greater, about 70% or greater, about 80% orgreater, about 90% or greater or about 100% or greater.

In another embodiment, active agents are administered at atherapeutically effective dosage sufficient to decrease the ratio ofAβ42:Aβ40 in the CSF or plasma of a subject by about 15% or more, about20% or more, about 25% or more, about 30% or more, about 35% or more,about 40% or more, about 45% or more, or about 50% or more.

In another embodiment, active agents are administered at atherapeutically effective dosage sufficient to lower levels of Aβ in theCSF or plasma of a subject by about 15% or more, about 25% or more,about 35% or more, about 45% or more, about 55% or more, about 75% ormore, or about 95% or more.

Toxicity and therapeutic efficacy of such agents can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and can be expressed as the ratio LD50/ED50, andusually a larger therapeutic index is more efficacious. While agentsthat exhibit toxic side effects may be used, care should be taken todesign a delivery system that targets such agents to the site ofaffected tissue in order to minimize potential damage to unaffectedcells and, thereby, reduce side effects.

It is understood that appropriate doses depend upon a number of factorswithin the ken of the ordinarily skilled physician, veterinarian, orresearcher. The dose(s) of the small molecule will vary, for example,depending upon the identity, size, and condition of the subject orsample being treated, further depending upon the route by which thecomposition is to be administered, if applicable, and the effect whichthe practitioner desires the small molecule to have upon the subject.Exemplary doses include milligram or microgram amounts of the smallmolecule per kilogram of subject or sample weight (e.g., about 1microgram per kilogram to about 500 milligrams per kilogram, about 100micrograms per kilogram to about 5 milligrams per kilogram, or about 1microgram per kilogram to about 50 micrograms per kilogram). It isfurthermore understood that appropriate doses depend upon the potency.Such appropriate doses may be determined using the assays describedherein. When one or more of these compounds is to be administered to ananimal (e.g., a human), a physician, veterinarian, or researcher may,for example, prescribe a relatively low dose at first, subsequentlyincreasing the dose until an appropriate response is obtained. Inaddition, it is understood that the specific dose level for anyparticular animal subject will depend upon a variety of factorsincluding the activity of the specific agent employed, the age, bodyweight, general health, gender, and diet of the subject, the time ofadministration, the route of administration, the rate of excretion, andany drug combination.

The ability of an agent to inhibit amyloid deposition can be evaluatedin an animal model system that may be predictive of efficacy ininhibiting amyloid deposition in human diseases, such as a transgenicmouse expressing human APP or other relevant animal models where Aβdeposition is seen or for example in an animal model of AA amyloidosis.Likewise, the ability of an agent to prevent or reduce cognitiveimpairment in a model system may be indicative of efficacy in humans.Alternatively, the ability of an agent can be evaluated by examining theability of the agent to inhibit amyloid fibril formation in vitro, e.g.,using a fibrillogenesis assay such as that described herein, including aThT, CD, or EM assay. Also the binding of an agent to amyloid fibrilsmay be measured using a MS assay as described herein. The ability of theagent to protect cells from amyloid induced toxicity is determined invitro using biochemical assays to determine percent cell death inducedby amyloid protein. The ability of an agent to modulate renal functionmay also be evaluated in an appropriate animal model system.

The therapeutic agent of the invention may be also be administered exvivo to inhibit amyloid deposition or treat certain amyloid-relateddiseases, such as β₂M amyloidosis and other amyloidoses related todialysis. Ex vivo administration of the therapeutic agents of theinvention can be accomplished by contacting a body fluid (e.g., blood,plasma, etc.) with a therapeutic compound of the invention such that thetherapeutic compound is capable of performing its intended function andadministering the body fluid to the subject. The therapeutic compound ofthe invention may perform its function ex vivo (e.g., dialysis filter),in vivo (e.g., administered with the body fluid), or both. For example,a therapeutic compound of the invention may be used to reduce plasma β₂Mlevels and/or maintain β₂M in its soluble form ex vivo, in vivo, orboth.

Prodrugs

The present invention is also related to prodrugs of the agents of theFormulae disclosed herein. Prodrugs are agents which are converted invivo to active forms (see, e.g., R. B. Silverman, 1992, “The OrganicChemistry of Drug Design and Drug Action,” Academic Press, Chp. 8).Prodrugs can be used to alter the biodistribution (e.g., to allow agentswhich would not typically enter the reactive site of the protease) orthe pharmacokinetics for a particular agent. For example, a carboxylicacid group, can be esterified, e.g., with a methyl group or an ethylgroup to yield an ester. When the ester is administered to a subject,the ester is cleaved, enzymatically or non-enzymatically, reductively,oxidatively, or hydrolytically, to reveal the anionic group. An anionicgroup can be esterified with moieties (e.g., acyloxymethyl esters) whichare cleaved to reveal an intermediate agent which subsequentlydecomposes to yield the active agent. The prodrug moieties may bemetabolized in vivo by esterases or by other mechanisms to carboxylicacids.

Examples of prodrugs and their uses are well known in the art (see,e.g., Berge, et al., “Pharmaceutical Salts”, J. Pharm. Sci. 66, 1-19(1977)). The prodrugs can be prepared in situ during the final isolationand purification of the agents, or by separately reacting the purifiedagent in its free acid form with a suitable derivatizing agent.Carboxylic acids can be converted into esters via treatment with analcohol in the presence of a catalyst.

Examples of cleavable carboxylic acid prodrug moieties includesubstituted and unsubstituted, branched or unbranched lower alkyl estermoieties, (e.g., ethyl esters, propyl esters, butyl esters, pentylesters, cyclopentyl esters, hexyl esters, cyclohexyl esters), loweralkenyl esters, dilower alkyl-amino lower-alkyl esters (e.g.,dimethylaminoethyl ester), acylamino lower alkyl esters, acyloxy loweralkyl esters (e.g., pivaloyloxymethyl ester), aryl esters (phenylester), aryl-lower alkyl esters (e.g., benzyl ester), substituted (e.g.,with methyl, halo, or methoxy substituents) aryl and aryl-lower alkylesters, amides, lower-alkyl amides, dilower alkyl amides, and hydroxyamides.

Pharmaceutically Acceptable Salts

Certain embodiments of the present agents can contain a basic functionalgroup, such as amino or alkylamino, and are, thus, capable of formingpharmaceutically acceptable salts with pharmaceutically acceptableacids. The term “pharmaceutically acceptable salts” in this respect,refers to the relatively non-toxic, inorganic and organic acid additionsalts of agents of the present invention. These salts can be prepared insitu during the final isolation and purification of the agents of theinvention, or by separately reacting a purified agent of the inventionin its free base form with a suitable organic or inorganic acid, andisolating the salt thus formed.

Representative salts include the hydrohalide (including hydrobromide andhydrochloride), sulfate, bisulfate, phosphate, nitrate, acetate,valerate, oleate, palmitate, stearate, laurate, benzoate, lactate,phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate,napthylate, mesylate, glucoheptonate, lactobionate,2-hydroxyethanesulfonate, and laurylsulphonate salts and the like. See,e.g., Berge et al., “Pharmaceutical Salts”, J. Pharm. Sci. 66, 1-19(1977).

In other cases, the agents of the present invention may contain one ormore acidic functional groups and, thus, are capable of formingpharmaceutically acceptable salts with pharmaceutically acceptablebases. The term “pharmaceutically acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of agents of the present invention.

These salts can likewise be prepared in situ during the final isolationand purification of the agents, or by separately reacting the purifiedagent in its free acid form with a suitable base, such as the hydroxide,carbonate or bicarbonate of a pharmaceutically acceptable metal cation,with ammonia, or with a pharmaceutically acceptable organic primary,secondary or tertiary amine. Representative alkali or alkaline earthsalts include the lithium, sodium, potassium, calcium, magnesium, andaluminum salts and the like. Representative organic amines useful forthe formation of base addition salts include ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.

“Pharmaceutically acceptable salts” also includes, for example,derivatives of agents modified by making acid or base salts thereof, asdescribed further below and elsewhere in the present application.Examples of pharmaceutically acceptable salts include mineral or organicacid salts of basic residues such as amines; and alkali or organic saltsof acidic residues such as carboxylic acids. Pharmaceutically acceptablesalts include the conventional non-toxic salts or the quaternaryammonium salts of the parent agent formed, for example, from non-toxicinorganic or organic acids. Such conventional non-toxic salts includethose derived from inorganic acids such as hydrochloric, hydrobromic,sulfuric, sulfamic, phosphoric, and nitric acid; and the salts preparedfrom organic acids such as acetic, propionic, succinic, glycolic,stearic, lactic, malic, tartaric, citric, ascorbic, palmoic, maleic,hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, and isethionic acid. Pharmaceutically acceptablesalts may be synthesized from the parent agent which contains a basic oracidic moiety by conventional chemical methods. Generally, such saltsmay be prepared by reacting the free acid or base forms of these agentswith a stoichiometric amount of the appropriate base or acid in water orin an organic solvent, or in a mixture of the two.

All acid, salt, base, and other ionic and non-ionic forms of thecompounds described are included as compounds of the invention. Forexample, if a compound is shown as an acid herein, the salt forms of thecompound are also included. Likewise, if a compound is shown as a salt,the acid and/or basic forms are also included.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures, embodiments, claims, and examples described herein.Such equivalents are considered to be within the scope of this inventionand covered by the claims appended hereto. The contents of allreferences, issued patents, and published patent applications citedthroughout this application are hereby incorporated by reference. Theinvention is further illustrated by the following examples, which shouldnot be construed as further limiting.

EXAMPLES Binding and Antifibrillogenic Assays

The test compounds were synthesized and screened by mass spectrometry(“MS”) assays, except for selected compounds which were purchased from acommercial source. The MS assay gives data on the ability of compoundsto bind to proteins, in this example, to β-amyloid and IAPP.

In the MS assay for Aβ40, samples were prepared as aqueous solutions(adding 20% ethanol if necessary to solubilize in water), 200 μM of atest compound and 20 μM of solubilized Aβ40, or 400 μM of a testcompound and 40 μM of solubilized Aβ40. The pH value of each sample wasadjusted to 7.4 (±0.2) by addition of 0.1% aqueous sodium hydroxide. Thesolutions were then analyzed by electrospray ionization massspectrometry using a Waters ZQ 4000 mass spectrometer. Samples wereintroduced by direct infusion at a flow-rate of 25 μL/min within 2 hr.after sample preparation. The source temperature was kept at 70° C. andthe cone voltage was 20 V for all the analysis. Data were processedusing Masslynx 3.5 software. The MS assay gives data on the ability ofcompounds to bind to soluble Aβ, whereas the ThT, EM and CD assays givedata on inhibition of fibrillogenesis. The results from the assay forbinding to Aβ are summarized in Table 3. In Table 3, a blank box meansthat a value was not determined for that compound in that assay.

The assay for IAPP was conducted under the same conditions except that200 μM or 100 μM of test compound and 20 μM of solubilized IAPP wereemployed. The key below describes the codes used in Table 3A to quantifythe binding based on the intensity of the absorption.

Key to Table 3A Aβ1-40 Code 400 μM 200 μM Strong Binding +++ 90-100% 60-100%  Moderate Binding ++ 70-89% 30-69% Weak Binding + 45-59% 20-44%Little/no − 20-39% 20-39% detectable binding IAPP 200 μM 100 μM (20%EtOH) (20% EtOH) Strong Binding +++ >75% >50% Moderate Binding ++ 40-75%30-50% Weak Binding + 20-40% 15-30% Little/no −  0-20%  0-15% detectablebinding

TABLE 3A Relative Binding Affinities of Compounds of the Invention MSbinding ID IAPP Aβ1-40 B ++ + C + D + E +++ + F +++ + G ++ + H +++ + I+++ ++ J +++ + K ++ − L ++ + M ++ − N + − P ++ − Q − − AC ++ + AD + AE +AF +++ + AG ++ ++ AH ++ + AK +++ ++ AL +++ ++ AM ++ + AW ++ ++ AX + ++AY ++ +++ AZ +++ ++ BA ++ ++ BB +++ +++ BC − + BV + +++ BW ++ +++ BX +++++ BY ++ +++ BZ +++ CC − ++ CD +++ ++ CE + +++ CG ++ +++ CH +++ +++CI + +++ CJ ++ +++ CK +++ +++ CM + CV + +++ CY +++ +++ DC ++ +++ DD +++DE + − DF +++ − DG − + DH + ++ DI − − DJ +++ − DK ++ +++ DL − + DM + ++DN + ++ DO ++ +++ DP − + DQ − + DR + + DS + + DT + + DU +++ +++ DV ++++++ DW +++ ++ DX +++ +++ DY ++ +++ DZ +++ +++ EA + ++ EB +++ +++ EC +++ED ++ +++ EE ++ +++ EF − + EG +++ +++ EH +++ +++ EI − − EJ + ++ EK ++++++ EL + ++ EN + + EO − + EP ++ + EQ − + ER ++ ES +++ +++ ET +++ EV +++EW +++ EY +++ +++ EZ +++ +++ FA ++ +++ FH − − FO − − FP +++ ++ FQ ++ +FR +++ ++ FS +++ +++ FT − − FU ++ ++ FV − − FW + − FX +++ +++ FY +++ +++FZ ++ + GA + − GB +++ + GC − − GD +++ ++ GE ++ GF + GH ++ + GI ++ + GJ++ ++ GK + + GL ++ ++ GM ++ − GN − − GO − − GP + − GQ − − GR − − GS ++ +GT + − GU ++ + GZ + − HA − − HB − − HC ++ + HD − − HE + − HF ++ − HG + +HI ++ HJ ++ − HK − − NG ++ NH + NI + NJ +++ NK + NL +++

Compounds in Table 3B were tested under the following conditions: 30 μMAβ peptide, 150 μM compound; 60-100% binding=(+++), 40-59% binding=(++),20-39% binding=(+), <20%=(−), blank=not determined.

TABLE 3B Relative Binding Affinities of Compounds of the Invention MSbinding ID IAPP Aβ1-40 AI +++ FN ++ IY +++ ++ JH +++ ++ JI +++ +++ JP+++ JQ +++ JR +++ ++ JV ++ KB +++ KH ++ KJ +++ KM +++ KN ++ KS +++ KT ++LK ++ LN +++

Compounds in Table 3C were tested under the following conditions 20 μMAβ peptide, 100 μM compound; 75-100% binding=(+++), 40-74% binding=(++),20-39% binding=(+), <20%=(−), blank=not determined.

TABLE 3C Relative Binding Affinities of Compounds of the Invention MSbinding ID IAPP Aβ1-40 IW +++ ++ IX +++ ++ IZ +++ − JA ++ + JB + + JC +JD ++ + JE ++ − JF ++ + JG +++ JJ +++ ++ JK + − JL ++ ++ JM + − JN + ++JO ++ + JS + JT + JU ++ JW ++ JX ++ JY +++ JZ +++ KA ++ KI + KK +++ KL++ KQ + LD ++ LE ++ LF + LG ++ LH + LI +++ LJ +++ LL ++ LM +++ LQ +++

Compounds in Table 3D were tested under the following conditions: 40 μMAβ peptide, 400 μM compound; 75-100% binding=(+++), 40-74% binding=(++),20-39% binding=(+), <20%=(−), blank=not determined.

TABLE 3D Relative Binding Affinities of Compounds of the Invention MSbinding ID IAPP Aβ1-40 S ++ − HL + ++ HM − + HN − HP − HQ + HR − ++ HS −HT + ++ HU + +++ HV + ++ HW − HX − HY − HZ − IA + +++ IB + IF − IG +++++ IH + II − IJ + IK + IL + IM − IN + IP + IR + IS + IT + IU − KV +KW + KX + KY + LA − LC − LP +++Effects of Short Term Treatment in Adult Transgenic CRND8 MiceOverexpressing βAPP

Transgenic mice, TgCRND8, expressing the human amyloid precursor protein(hAPP) develop a pathology resembling Alzheimer's disease. Inparticular, high levels of Aβ40 and Aβ 42 have been documented in theplasma and the brain of these animals at 8-9 weeks of age, followed byearly accumulation of amyloid plaques similar to the senile plaquesobserved in AD patients. These animals also display progressivecognitive deficits that parallel the appearance of degenerative changes.See, e.g., (Chishti, et al., J. Biol. Chem. 276, 21562-70 (2001).

The short term therapeutic effect of 19 compounds of the invention wasstudied. These compounds were administered over a 14 or 28 day period atthe end of which the levels of Aβ peptides in the plasma and brain ofTgCRND8 animals were determined.

Methods

Male and female transgenic mice from the 3^(rd) and 4^(th) B6C3F1generations were used in this example and given daily subcutaneous ororal administrations of one of a series of compounds for 14 or 28 days.The following abbreviations are used to designate these animals from the3^(rd) and 4th generation backcross in the present protocol:TgCRND8-2.B6C3F1(N₃); TgCRND8-2.B6C3F1(N₄).

Baseline animals (Group 1) consisted of naive TgCRND8-2. B6C3F1(N₃) at11±1 weeks of age. These mice were used to determine the Aβ levels inthe plasma and brain of naive transgenic animals at the initiation oftreatment.

Starting at 11 weeks of age (±1 week) animals received dailyadministration of their respective treatment for a period of 14 or 28days (groups 2-21), at a dose of 250 mg/kg at 10 ml/kg or of vehicleonly (water; group 2) or 1% methyl cellulose only (group 21). The routeof administration was subcutaneous for water-soluble compounds and oralfor compounds solubilized in methylcellulose 1% (MC 1%). At the end ofthe treatment periods, plasma and perfused brains were collected forquantification of Aβ levels.

TABLE 4 Test System Species: Mouse Strain: TgCRND8-2.B6C3F1(N₃) & (N₄)Genotype: hAPP+/− Gender: Male and Female Age at Day 1: 11 ± 1 weeksBody Weight 10 to 30 g at Day 1: Number of Baseline: 8 Animals/Group atDay 1: Vehicle and 12-15 Treated: Suppliers: TgCRND8-2 founders wereobtained from the Centre for Research in Neurodegenerative Diseases,University of Toronto. The inbred B6C3F1 were obtained from CharlesRiver (Quebec, Canada).

The mice used in this study were derived from a breeding colony atInstitut Armand Frappier, and were well-acclimated to the animalfacility environment prior to initiation of the study Animals wereassigned, according to age and gender, into the following experimentalgroups:

TABLE 5 Groups of Mice Daily Dose Duration of Treatment Group No.Treatment (mg/kg) (days) 1 Baseline NA NA 2 Water NA 14 & 28 4 BY 250 14& 28 6 CV 250 14 & 28 12 CY NA 14 & 28 15 BW 250 14 & 28 16 BZ 250 14 &28 18 BX 250 14 & 28 20 DC 250 14 & 28 21 Methylcellulose 1% 100 14 & 2822 DD 250 14 & 28 23 DH 250 14 & 28 24 DM 250 14 & 28 25 DX 250 14 & 2826 DY 250 14 & 28 27 DZ 250 14 & 28 28 ED 250 14 & 28 29 EG 250 14 & 28Animal Health Monitoring

All animals were examined daily for signs of ill health when handled inthe morning for their daily treatment and twice a day for mortalitychecks (once daily during weekends and holidays). Detailed examinationswere performed on the treatment initiation, weekly during the study, andonce before terminal procedures. More frequent observations wereundertaken when considered appropriate. Death and all individualclinical signs were individually recorded. Individual body weights wererecorded at randomization, once weekly during the study, and once beforeterminal procedures.

Sample Collection

At 11±1 weeks of age for the Baseline group, and at the end of thetreatment period (14 or 28 days) for Groups 2 to 21, at 24 hours afterthe last compound administration animals were sacrificed and samplescollected. An approximate blood volume of 500 μl was collected from theorbital sinus and kept on ice until centrifugation at 4° C. at a minimumspeed of 3,000 rpm for 10 minutes. Plasma samples were immediatelyfrozen and stored at −80° C. pending analysis. The brains were removed,frozen, and stored at −80° C. awaiting analysis.

Measurements of Aβ Levels

Brains were weighted frozen and homogenized with 4 volumes of ice cold50 mM Tris-Cl pH 8.0 buffer with protease inhibitor cocktail (4 mL ofbuffer for 1 g of wet brain). Samples were spun at 15000 g for 20minutes and the supernatants were transferred to fresh tubes. Onehundred fifty (150) μl from each supernatant were mixed with 250 μl of8M guanidine-HCL/50 mM Tris-HCL pH 8.0 (ratio of 0.6 vol supernatant: 1vol 8M guanidium/Tris-HCL 50 mM pH 8.0) and 400 μL 5 Mguanidium/Tris-HCL 50 mM pH8.0 were added. The tubes were vortexed for30 seconds and frozen at −80° C. In parallel, pellets were treated with7 volumes of 5 M guanidine-HCL/50 mM Tris-HCL pH 8.0 (7 mL of guanidinefor 1 g of wet brain), vortexed for 30 seconds and frozen at −80° C.Samples were thawed at room temperature, sonicated at 80° C. for 15minutes and frozen again. This cycle was repeated 3 times to ensurehomogeneity and samples were returned to −80° C. pending analysis.

Aβ levels were evaluated in plasma and brain samples by ELISA usingHuman Aβ40 and Aβ42 Fluorometric ELISA kits from Biosource (Cat. No.89-344 and 89-348) according to manufacturer's recommended procedures.In short, samples were thawed at room temperature, sonicated for 5minutes at 80° C. (sonication for brain homogenates; no sonication forplasma samples) and kept on ice. Aβ peptides were captured using 100 μlof the diluted samples to the plate and incubated without shaking at 4°C. overnight. The samples were aspirated and the wells were rinsed 4times with wash buffer obtained from the Biosource ELISA kit. Theanti-Aβ40 or anti-Aβ42 rabbit polyclonal antiserum (specific for theAβ40 or Aβ42 peptide) was added (100 μl) and the plate was incubated atroom temperature for 2 hours with shaking. The wells were aspirated andwashed 4 times before adding 100 μl of the alkaline phosphatase labeledanti-rabbit antibody and incubating at room temperature for 2 hours withshaking. The plates were then rinsed 5 times and the fluorescentsubstrate (100 μl) was added to the plate. The plate was incubated for35 minutes at room temperature and the plate was read using a titerplate reader at an excitation wavelength of 460 nm and emission at 560nm

Compounds were scored based on their ability to modulate levels of Aβpeptides in the plasma and the cerebral soluble/insoluble levels in thebrain. Levels of Aβ observed in the plasma and brain of treated animalswere normalized using values from vehicle-treated (water) ormethylcellulose-treated control groups and ranked according to thestrength of the pharmacological effect. Results are shown in Tables 3 to11. Increases in the levels of Aβ peptides are indicated using “+”symbols, while decreases in the levels of Aβ peptides are indicatedusing “−” symbols. The strongest effects are recorded as “+++” or “− −−” while the weakest are shown as “+” or “−”.

Specifically, increases in the levels of Aβ (relative to untreatedcontrol) of 20 to 39% are scored as “+”; increases of 40 to 69% arescored as “++”; and increases of 70% or higher are scored as “+ + +”.Decreases in the levels of Aβ of 5 to 19% are scored as “−”; decreasesof 20 to 38% are scored as “− −”; and decreases of 39% or more arescored as “− − −”.

The data are presented in Tables 6-11. Treatment with these compoundsafter 14 and/or 28 days resulted in a significant change in the cerebrallevels of Aβ40 and/or Aβ42 (Tables 8-11). Furthermore, treatment withthese compounds, for instance, Compound BX(3-(t-butyl)amino-1-propanesulfonic acid), resulted after 14 and 28 days(Tables 6-7) in a significant increase in the levels of Aβ peptides inthe plasma. This suggests that some of these compounds may act by aperipheral sink effect, sequestering Aβ peptides in the plasma andthereby facilitating their clearance from the CNS as previouslysuggested for treatment by passive immunization using anti-Aβ monoclonalantibody m266 (DeMattos et al., Science 295(5563):2264-7).

The tables show levels of Aβ peptides in the plasma and brain of TgCRND8mice treated for 14 and 28 days with compounds of the invention.

Tables 6A and 6C show the data from Day 14 and Day 28 for the PlasmaVehicle group, respectively. Tables 6B and 7 show the data for thePlasma Methylcellulose group on Days 14 and 28, respectively. Tables 8and 10 show the data on Days 14 and 28 for the Brain homogenate vehiclegroup, respectively. Tables 9 and 11 show the data for brain homogenatefor the Methylcellulose group on Days 14 and 28, respectively.

TABLE 6A Plasma Vehicle Group, Day 14 Treatment Aβ40 Change Aβ42 ChangeBY + + CV + ++ DC ++ ++ BX +++ ++

TABLE 6B Plasma Methylcellulose Group, Day 14 Treatment Aβ40 Change Aβ42Change BZ + BW CY

TABLE 6C Plasma Vehicle Group, Day 28 Treatment Aβ40 Change Aβ42 ChangeBY CV ++ ++ DC ++ BX +++

TABLE 7 Plasma Methylcellulose Group, Day 28 Treatment Aβ40 Change Aβ42Change BZ ++ ++ BW + CY +

TABLE 8 Brain Homogenate Vehicle Group, Day 14 Aβ40 Change Aβ42 ChangeTreatment Soluble Insoluble Soluble Insoluble BY +++ +++ +++ CV** − DC−− + −− BX − −−− −− −−− DD − DX −− − DY −− −− DZ −− EG − −− −− −− DH −−−−− −−− DM + − − ED − + **The effect of this compound in the brain hasonly been tested on its ability to modulate the total levels of Aβ40 andAβ42 peptides rather than measuring soluble and insoluble levelsindependently.

TABLE 9 Brain Homogenate Methylcellulose Group, Day 14 Aβ40 Change Aβ42Change Treatment Soluble Insoluble Soluble Insoluble BZ −−− −− BW −−− −−−−− CY − +++ ++

TABLE 10 Brain Homogenate Vehicle Group, Day 28 Aβ40 Change Aβ42 ChangeTreatment Soluble Insoluble Soluble Insoluble BY + +++ +++ CV** ++ +++DC − + ++ +++ BX −−− −−− −− − DD − DX −− − −− DY −−− − −− DZ − −− −− −−EG −− −− DH − − DM − −− −− −− ED − −− − − **The effect of this compoundin the brain has only been tested on its ability to modulate the totallevels of Aβ40 and 42 peptides rather than measuring soluble andinsoluble levels independently.

TABLE 11 Brain Homogenate Methylcellulose Group, Day 28 Aβ40 Change Aβ42Change Treatment Soluble Insoluble Soluble Insoluble BZ −− −− −− BW − −− −− CY ++ +++ +++ +Effects of Long Term Treatment in Adult Transgenic CRND8 MiceOverexpressing βAPP

Transgenic mice, TgCRND8, as those used in the short term treatment,overexpress a human APP gene with the Swedish and Indiana mutationsleading to the production of high levels of the amyloid peptides and tothe development of an early-onset, aggressive development of brainamyloidosis. The high levels of Aβ peptides and the relativeoverabundance of Aβ₄₂ compared to Aβ₄₀ are believed to be associatedwith the severe and early degenerative pathology observed. The patternof amyloid deposition, presence of dystrophic neuritis, and cognitivedeficit has been well documented in this transgenic mouse line. Thelevels of Aβ peptides in the brain of these mice increase dramaticallyas the animals age. While the total amyloid peptide levels increase from˜1.6×10⁵ pg/g of brain to ˜3.8×10⁶ between 9 and 17 weeks of age.

While the early deposition of amyloid in this model allows the rapidtesting of compounds in a relatively short time frame, the aggressivityof this model and the high levels of Aβ peptides renders therapeuticassessment in the longer term a more difficult task.

The long-term therapeutic effects of compounds of the present inventionon cerebral amyloid deposition and β-amyloid (Aβ) levels in the plasmaand in the brains of transgenic mice, TgCRND8, expressing the humanamyloid precursor protein (hAPP) was studied. These compounds wereadministered over an 8 or 16 week period at the end of which the levelsof Aβ peptides in the plasma and brain of TgCRND8 animals weredetermined. The goal of this study was to evaluate the efficacy of thecompounds at modulating the progression of the amyloidogenic process inthe brain and in the plasma of a transgenic mouse model of Alzheimer'sdisease (AD)

Methods

The mice used in the study consisted of animals bearing one copy of thehAPP gene (+/−) from the 2^(nd) and 3^(rd) generation progenies (N₂ andN₃) derived from backcrosses from TgCRND8.FVB(N₂)AJ(N₃) with B6AF1/Jhybrid animals.N₁=TgCRND8.FVB(N₂)AJ(N₃)×B6AF1/JN₂=TgCRND8.FVB(N₂)AJ(N₃).B6AF1/J(N₁)×B6AF1/JN₃=TgCRND8.FVB(N₂)AJ(N₃).B6AF1/J(N₂)×B6AF1/a

The following abbreviations are used to designate these animals in thepresent study: TgCRND8.B6AF1/J(N₂); TgCRND8.B6AF1/J(N₃). Male and femaletransgenic mice were given daily subcutaneous (compound BX) or oral(compounds BW and BZ) administrations of the appropriate compounds for 8or 16 weeks.

Baseline animals consisted of 9±1 week old naive TgCRND8.B6AF1/J animalsfrom the 2^(nd) and 3^(rd) generations. These mice were used todetermine the extent of cerebral amyloid deposits and Aβ levels in theplasma and brain of naive transgenic animals at the initiation oftreatment.

Starting at 9 weeks of age (±1 week) animals received dailyadministration of their respective treatment for a period of 8 or 16weeks, at a dose of 30 or 100 mg/kg at 10 ml/kg. The route ofadministration was subcutaneous for water-soluble compounds (CompoundBX) and oral for compounds solubilized in methylcellulose 1% (MC 1%)(Compounds BW and BZ). At the end of the treatment periods, plasma andperfused brains were collected for quantification of Aβ levels.

Animal health was monitored, samples were collected and Aβ levels weremeasured as described above in the short term treatment study. Compoundswere scored based on their ability to modulate levels of Aβ peptides inthe plasma and the cerebral soluble/insoluble levels in the brain.Levels of Aβ observed in the plasma and brain of treated animals werecompared to that of vehicle-treated (water) or methylcellulose-treatedcontrol groups and ranked according to the strength of thepharmacological effect. Results are shown in Table 12. Increases in thelevels of Aβ peptides are indicated using “+” symbols, while decreasesin the levels of Aβ peptides are indicated using “−” symbols. Thestrongest effects are recorded as “+++” or “−−−” while the weakest areshown as “+” or “−”.

Specifically, increases in the levels of Aβ (relative to vehicle treatedcontrol) of 5 to 14% are scored as “+”; increases of 15 to 29% arescored as “++”; and increases of 30% or higher are scored as “+++”.Decreases in the levels of Aβ of 5 to 14% are scored as “−”; decreasesof 15 to 29% are scored as “−−”; and decreases of 30% or more are scoredas “−−−”. Additionally, changes of 4% or less in either direction arescored as “0”.

Table 12 shows levels of Aβ peptides in the plasma and brain of TgCRND8mice treated for 8 and 16 weeks with compounds of the invention.Treatment with these compounds after 8 and/or 16 weeks in many casesresulted in a change in the levels of Aβ and/or Aβ₄₂ in the plasmaand/or brain. For example, administration of compound BX generallyresulted in a dramatic decrease in the amount of Aβ in the brain at both8 and 16 weeks. Compound BW also resulted in a dramatic decrease inbrain and plasma levels of Aβ at 8 weeks and plasma levels at 16 weeks.

For the ThioS studies, the plaques in the brains of the mice werequantified as follows. Mice were transcardially perfused with salinesolution. Brains were dissected out and separated in 2 hemispheres. Theleft hemisphere was immersed in freshly-prepared 4% paraformaldehyde for3 hrs at 4° C., then transferred into 30% sucrose at 4° C. Whencryoprotection was achieved (24-48 hours), brains were frozen inisopentane at −45° C. and subsequently stored at −80° C. untilsectioning. Coronal 40 μm-thick sections were performed, and stainedwith thioflavin S (1% solution in water) for 8 min After differentiationof the thioflavin S staining, sections were counterstained withhematoxylin for 5 minutes. Two sets of pictures were capturedsimultaneously. A first set of pictures was captured under brightfieldillumination to obtain morphological details of the section; a secondset of pictures was captured under a green, specific, fluorescent filter(fluorescein filter, Ex 495 nm, Em 525 nm). Image analysis to quantifythe number of plaques and the area occupied by these plaques wasperformed using Image Pro Plus software (Media Cybernetics, MD, USA).

The data from the histological ThioS studies is summarized in Table 13.Increases in the areas and numbers of plaques are indicated using “+”symbols, while decreases in the areas and numbers of the plaques areindicated using “−” symbols. Preliminary histochemical experiments usingThioS staining of brain sections indicated that both the number ofplaques and the area occupied by the plaques were decreased in micetreated with 30 mg/kg of compound BX.

Specifically, increases in the areas and numbers of plaques (relative tovehicle treated control) of 10 to 19.99% are scored as “+”. Decreases inthe areas and numbers of plaques of 10 to 19.99% are scored as “−”.Additionally, changes of 9.99% or less in either direction are scored as“0”.

TABLE 12 Effects of Compounds BX, BW and BZ on levels of Aβ in plasmaand brain Brain Dose Timepoint Plasma Abeta40 Abeta42 Compound (mg/kg)(weeks) Abeta40 Abeta42 soluble insoluble soluble insoluble BX 30  8wks + + −−− −−− −−− −− BX 100  8 wks ++ +++ + ++ + 0 BX 30 16 wks − − −−−−− 0 − BX 100 16 wks − 0 − −−− 0 −− BW 30  8 wks −−− −−− − −− −− 0 BW100  8 wks − − −− −−− −− −−− BW 30 16 wks −− −− + ++ − + BW 100 16 wks −− ++ + + ++ BZ 30  8 wks 0 0 0 −− 0 −−− BZ 100  8 wks ++ +++ ++ 0 0 − BZ30 16 wks 0 + 0 + + 0 BZ 100 16 wks ++ ++ −− 0 − +

TABLE 13 Histological effects of compounds BW and BX on numbers ofplaques and areas occupied by plaques ThioS Sites Analyzed SurfaceChange in Change in Com- Dose Timepoint Area Plaque Plaque pound (mg/kg)(weeks) (μm²) Number Area BX 30 16 wks 7,773,230 − − BX 100 16 wks7,803,230 0 + BW 30 16 wks 7,563,737 0 0 BW 100 16 wks 7,812,844 − 0Evaluation of Compounds Binding to NAC Peptide by Mass Spectrometry

Recent findings have demonstrated that a high percentage of AlzheimerDisease (AD) patients also form Lewy bodies, most abundantly in theamygdala (Hamilton. 2000 Brain Pathol, 10:378; Mukaetova-Ladinska, etal. 2000. J Neuropathol Exp Neurol 59:408). Interestingly, the highlyhydrophobic non-amyloid component (NAC) region of α-synuclein has alsobeen described as the second most abundant component of amyloid plaquesin the brain of AD patients, after. Alpha-synuclein has been shown toform fibrils in vitro. Furthermore it binds to Aβ and promotes itsaggregation (Yoshimoto, et al. 1995. Proc Natl Acad Sci USA 92:9141). Itwas in fact originally identified as the precursor of the non-amyloidbeta (Aβ) component (NAD) of AD plaques (Ueda, et al. 1993. Proc NatlAcad Sci USA 90:11282; Iwai. 2000. Biochem Biophys Acta 1502:95;Masliah, et al. 1996. Am J Pathol 148:201). NAC is a 35 amino acid longpeptide with highly hydrophobic stretches which can self-aggregate andform fibrils in vitro. Moreover, these fibrils can efficiently seed theformation of Aβ fibrils in vitro (Han, et al. 1995. Chem. Biol. 2:163-169; Iwai, et al. 1995. Biochemistry 34:10139). It is in factthrough its NAC domain that alpha-synuclein retains its fibrillogenicproperties. Modulating the properties of NAC or targeting NAC with thecompounds of the invention could therefore be a valid therapeutic avenueto inhibit the formation of protein aggregates and inclusions associatedwith alpha-synucleopathies, as well as the formation of aggregatesbetween the beta-amyloid peptide and NAC of alpha-synuclein.

The ability of the compounds of the present invention to bind to NACpeptide in aqueous solution was evaluated. The binding abilitycorrelates to the intensities of the peptide-compound complex peaksobserved by the Electrospray Mass Spectrum. Millipore distilleddeionized water was used to prepare all aqueous solutions. For pHdetermination a Beckman Φ36 pH meter fitted with a Corning Semi-MicroCombination pH Electrode was employed.

NAC (MW 3260.6 Da) at 20 μM was first analyzed at pH 7.40 and the usualsodium clusters was observed at +2, +3 and +4 at m/z 1335.5, 1116.7 and843.4 respectively. The optimal cone voltage was determined to be 20V.

Mass spectrometry—Mass spectrometric analysis was performed using aWaters ZQ 4000 mass spectrometer equipped with a Waters 2795 samplemanager. MassLynx 4.0 (earlier by MassLynx 3.5) was used for dataprocessing and analysis. Test compounds were mixed with disaggregatedpeptides in aqueous media (6.6% EtOH) at a 5:1 ratio (20 μM NAC: 100 μMof test compound or 40 μM NAC:200 μM of test compound). The pH of themixture was adjusted to 7.4 (±0.2) using 0.1% NaOH (3-5 μL).Periodically, NAC peptide solution at 20 μM or 40 μM was also preparedin the same fashion and run as control. The spectra were obtained byintroducing the solutions to the electrospray source by direct infusionusing a syringe pump at a flow rate of 25 μl/min, and scanning from 100to 2100 Da in the positive mode. The scan time was 0.9 sec per scan withan inter-scan delay of 0.1 sec and the run time was 5 min for eachsample. All the mass spectra were sum of 300 scans. The desolvation andsource temperature was 70° C. and the cone and capillary voltage weremaintained at 20 V and 3.2 kV respectively.

The total area under the peaks for the bound NAC-compound complexdivided by total area under the peaks for unbound NAC was determined foreach compound tested. The results are summarized in Table 14 below.

TABLE 14 NAC Peptide Binding Data Structure Binding Strength *NaO₃SCH₂(CH₂)₂CH₂SO₃Na − NaO₃SOCH₂CH₂CH₂OSO₃Na − NH₂CH₂CH₂OSO₃H −H₂NCH₂CH₂CH₂OSO₃Na ++ H₂NCH₂CH₂SO₃H +

++

++

+

+++

* +++ = Strong; when the total binding is 120% and higher ++ = Moderate;when the total binding is between 120% and 70% + = Weak; when the totalbinding is between 70% and 30% − = None; when the total binding isbetween 30% and 0%

The present invention also relates to novel compounds and the synthesisthereof. Accordingly, the following examples are presented to illustratehow some of those compounds may be prepared.

Synthesis of Compounds of the Invention

Preparation of 3-isopropylamino-1-propanesulfonic acid (Compound CG)

Isopropylamine (2.5 mL, 29 mmol) was added to a solution of 1,3-propanesultone (3.05 g, 25 mmol) in a mixture of dichloromethane/ether (40 mL,1:1). The mixture was heated at reflux for 3 hours. The reaction mixturewas cooled to room temperature and hexane (10 mL) was added. The solidmaterial was collected by filtration, rinsed with ether (10 mL), anddried in vacuo. Compound CG was obtained as a fine white powder (2.98 g,65.6% yield). m.p. 240-43° C. ¹H NMR (500 MHz, D₂O) δ 1.06 (d, J=6.3 Hz,6H), 1.86 (qt, J=7.6 Hz, 2H), 2.76 (t, J=7.6 Hz, 2H), 3.14 (t, J=7.8 Hz,2H), 3.13-3.21 (m, J=6.6 Hz, 1H). ¹³C NMR (125 MHz, D₂O) δ 18.2, 21.5,25.8, 43.4, 47.9, 50.8.

Preparation of 3-cyclopropylamino-1-propanesulfonic acid (Compound CI)

Cyclopropylamine (3.7 mL, 52 mmol) was added to a solution of1,3-propane sultone (6.9 g, 55.3 mmol) in THF (60 mL). The mixture washeated with an oil-bath at 42° C. for 2 hours. Stirring was difficultand part of the solid formed a crust above the stirred mixture. Themixture was heated at reflux for 1 hour, cooled to room temperature. Thesolid material was collected by filtration, dried in a vacuum oven for 2hours at 60° C. (4.95 g). The solid was recrystallized in methanol/water(35 mL/5 mL, v/v). The mixture was cooled in a fridge then the solid wascollected by filtration, rinsed with methanol (15 mL), air-dried for 15minutes, and further dried in a vacuum oven at 60° C. overnight.Compound CI was obtained as long, fine, white needles (3.74 g, 40%yield). m.p. 234-236° C. ¹H NMR (500 MHz, D₂O) δ 0.62-0.71 (m, 4H), 1.92(qt, J=7.6 Hz, 2H), 2.51-2.55 (m, J=3.7 Hz, 1H), 2.78 (d, J=7.3 Hz, 2H),3.09 (t, J=7.8 Hz, 2H). ¹³C NMR (125 MHz, D₂O) δ 3.0, 21.2, 30.0, 46.8,47.9. FT-IR (KBr) ν_(max) 3051, 1570, 1465, 1039

Preparation of 3-cyclopentylamino-1-propanesulfonic acid (Compound CJ)

Cyclopentylamine (3.95 mL, 40 mmol) was added to a solution of1,3-propane sultone (5.5 g, 45 mmol) in THF (80 mL). The mixture washeated at reflux with an oil-bath for 4 hours. Stirring was difficult,some acetone and ethanol were added to restore stirring. The mixture wascooled to room temperature. The solid was collected by filtration, driedin a vacuum oven for 1 hour at 60° C. (5.47 g). The solid material wasdissolved in methanol/water (35 mL/2.5 mL, v/v) at reflux. The mixturewas cooled slowly to room temperature overnight, and further cooled in afridge. The product was collected by filtration, rinsed with methanol(15 mL), air-dried for 15 minutes, and further dried in a vacuum oven at60° C. overnight. The product, Compound CJ, was obtained as long finewhite needles (4.79 g). A second crop was obtained from the combinedcrude and first recrystallization mother liquor (0.84 g). Both cropswere pure and were combined, total 5.63 g, 68% yield. m.p. 280-82° C. ¹HNMR (500 MHz, D₂O) δ 1.34-1.43 (m, 4H), 1.46-1.54 (m, 2H), 1.82-1.90 (m,4H), 2.76 (7, J=7.6 Hz, 2H), 2.95 (t, J=7.8 Hz, 2H), 3.35 (qt, J=7.2 Hz,1H). ¹³C NMR (125 MHz, D₂O) δ 21.5, 23.6, 29.3, 45.1, 47.9, 59.5. FT-IR(KBr) ν_(max) 3558, 3501, 2972, 1647, 1587, 1466

Preparation of 3-cycloheptylamino-1-propanesulfonic acid (Compound CK)

Cycloheptylamine (3.9 mL, 30 mmol) was added to a solution of1,3-propane sultone (4.1 g, 33 mmol) in THF (65 mL). The mixture washeated at reflux for 5 hours with a heating mantle. The mixture wascooled to room temperature and the solid was collected by filtration,and then dried in a vacuum oven for 1 hour at 60° C. (6.21 g). The solidmaterial was dissolved in methanol/water (30 mL/3 mL, v/v). The solutionwas cooled slowly to room temperature, and further cooled with anice-bath. The solid product was collected by filtration, rinsed withmethanol, air-dried for 15 minutes, and further dried in a vacuum ovenat 60° C. Compound CK was obtained as small white flakes (5.08 g, 72%yield). m.p. 341-43° C. ¹H NMR (500 MHz, D₂O) δ 1.21-1.42 (m, 8H),1.45-1.51 (m, 2H), 1.79-1.89 (m, 4H), 2.76 (7, J=7.3 Hz, 2H), 2.96 (t,J=7.8 Hz, 2H), 3.35 (m, J=4.6 Hz, 1H). ¹³C NMR (125 MHz, D₂O) δ 21.6,23.3, 27.3, 30.5, 43.6, 48.0, 59.6. FT-IR (KBr) ν_(max) 2924, 1615,1464, 1243.

Preparation of 3-benzyloxycarbonylamino-2-hydroxy-1-propanesulfonicacid, sodium salt (Compound AC)

3-Amino-2-hydroxy-1-propanesulfonic acid (15.51 g, 100 mmol) wasdissolved in water (150 mL) with the help of 1 equivalent of NaOH (4.08g). A solution of CBZ-OSuc (27.4 g, 110 mmol) in MeCN (300 mL) wasadded. After stiffing for 4 hours at room temperature, the solvent wasevaporated under reduced pressure. The wet cake (one equivalent inweight of water) was then suspended in acetone (400 mL) and heated underreflux for 20 minutes. The mixture was cooled to room temperature andthe solid was collected by filtration, washed with acetone and driedovernight in then vacuum oven at 40° C. Compound AC was obtained as awhite fine solid (28.85 g, 87.6 mmol, 88%). The ¹H and ¹³C NMR wereconsistent with the structure.

Preparation of 4-benzyloxycarbonylamino-1-butanesulfonic acid, sodiumsalt (Compound AD)

4-Amino-1-butanesulfonic acid sodium salt (0.516 g, 2.95 mmol wasdissolved in water for a final concentration of 0.5 M (slightly yellowsolution). A solution of CBZ-OSuc in CH₃CN (2 M, 1.55 mL, 3.1 mmol, 1.05eq.) was added. The reagent precipitated. A mixture of 1,4-dioxane andethanol was added until almost all of the solid was dissolved. After3.75 h, the solvent was evaporated under reduced pressure. The solid wasdried in vacuo over the weekends. The solid was then suspended inacetone and heated under reflux for 30 minutes. The mixture was cooledto room temperature and the solid was collected by filtration, washedwith acetone and dried overnight in vacuo. Compound AD was obtained as awhite fine solid (0.7610 g, 2.32 mmol, 78%). The ¹H NMR was consistentwith the structure.

Preparation of 3-benzyloxycarbonylamino-1-propanesulfonic acid sodiumsalt (Compound AE)

3-Amino-1-propanesulfonic acid sodium salt (1.09 g, 6.76 mmol) wasdissolved in water for a final concentration of 0.5 M. A solution ofCBZ-OSuc in CH₃CN (2 M, 3.55 mL, 7.1 mmol, 1.05 eq.) was added. Thereagent precipitated. A mixture of 1,4-dioxane and ethanol was addeduntil almost all the solid was dissolved. After 3.75 hours, the solventwas evaporated under reduced pressure. The solid was dried in vacuo overthe weekends. The solid was then suspended in acetone and heated underreflux for 30 minutes. The mixture was cooled to room temperature andthe solid was collected by filtration, washed with acetone and driedovernight in vacuo. Compound AE was obtained as a white fine solid (1.58g, 5.06 mmol, 75%). The ¹H NMR was consistent with the structure. 90%pure (10% mol of homotaurine).

Preparation of L-(N-Boc)-Phe-L-Phe-Homotaurine Isobutyl Ester

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Component 1: L-(N-Boc)-Phe-L-Phe

A solution of (Boc)₂O (800 mg, 3.5 mmol) in 1,4-dioxane (5 mL) was addedto a cold (0° C.) solution of di-L-Phe-Phe (1 g, 3.20 mmol) in1,4-dioxane (6 mL) and 1N NaOH (3.3 mL). The mixture was stirred at 0-5°C. for 2 hours. Another portion of (BOC)₂O (100 mg) was added and themixture was stirred for an additional 60 minutes at 0-5° C. then at roomtemperature for 30 minutes. The mixture was then evaporated to dryness.The solid was taken in a mixture of water/EtOAc and pH was adjusted to 2with 2N HCl. The aqueous layer was extracted 3 times with EtOAc. Thecombined organic layers were dried with brine and the solvent wasevaporated. Some solid was insoluble in a mixture of EtOAc/CHCl₃: it wasremoved by filtration. The desired N-Boc-L-Phe-L-Phe 1 was obtained as awhite foamy solid (913.7 mg, 2.215 mmol, 71% yield).

Component 2: 3-amino1-propanesulfonic acid isobutyl ester

Step 1: 3-azido-1-propanesulfonic acid, sodium salt (5)

A solution of 1,3-propane sultone 4 (6.12 g, 49.1 mmol) in acetone (30mL) was added to a mixture of sodium azide (3.22 g, 49.1 mmol) inwater/acetone (70 mL, 20 to 50). The clear solution was stirred at roomtemperature. The reaction was completed within 1 hour. The solvent wasremoved by evaporation under reduced pressure. The solid obtained wasrinsed with hot ether (50 mL) and then with ether at room temperature(150 ml). The solid was then dried in the vacuum oven at 40° C. forovernight. The title compound 5 was obtained as a white solid (8.69 g,46.4 mmol, 95% yield).

Step 2: 3-azido-1-propanesulfonyl chloride

3-Azidopropanesulfonic acid, sodium salt 5 (1.87 g, 10.0 mmol) wassuspended in dry benzene (20 mL), and PCl₅ (2.3 g, 10.5 mmol) was addedto the suspension. The mixture was stirred at room temperature for 30minutes, then at gentle reflux for about 1 hour. The benzene and P(O)Cl3were removed by evaporation under reduced pressure. Benzene was added tothe crude mixture and the solvent was removed again under reducedpressure. The residue was dried in vacuo. The dried residue wasdissolved in dichloromethane (anhydrous, 15 mL) and cooled at −10° C.using an ice/acetone bath.

Step 3: 3-azido-1-propanesulfonic acid, isobutyl ester

A solution of isobutanol (1.00 mL, 10.8 mmol) and 2,6-lutidine (1.3 mL,11.2 mmol) in dichloromethane (10 mL) was added slowly to the coldsulfonyl chloride solution. The mixture was stirred at −10° C. for 5minutes then at room temperature for 2 hours. The reaction was quenchedwith water and dichloromethane was added to extract the product. Theorganic layer was washed once with water, aqueous saturated NaHCO₃,brine, and then dried over magnesium sulfate. The solvent was removed byevaporation under reduced pressure and the residue was dried in vacuo.The remaining 2,6-lutidine hydrochloride was removed by washing theresidue with ether. The resulting oil (1.78 g) was then applied on flashcolumn (silica gel, EtOAc in Hexanes from 15% to 20%) to give afford thedesired ester as an oil (790 mg, 35%).

Step 4: 3-amino-1-propanesulfonic acid isobutyl ester

A solution of isobutyl 3-azidopropanesulfonate (1.13 g, 5.11 mmol) inisobutanol (10 mL) was added under H₂, via a cannula, to a suspension ofPd/C (10%, 200 mg) in isobutanol (4 mL) which had been saturated withH₂. The mixture was then stirred under H₂ (40 psi) at room temperatureovernight. The solid was then removed by filtration. The filtrate wasevaporated to dryness. And the residue was dried in vacuo. The titlecompound 2, homotaurine isobutyl ester, was obtained as brown oil (808.7mg, 81%).

Reaction of Component 1 with Component 2

HOBT (340 mg, 2.215 mmol) was added to a cold (0-5° C.) solution ofN-Boc-L-Phe-L-Phe 1 (913.7 mg, 2.215 mmol) and homotaurine isobutylester 2 (423 mg, 2.21 mmol) in dichloromethane (anhydrous, 30 mL). After5 minutes, a solution of 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimidemetho-p-toluenesulfonate (982 mg, 2.215 mmol) in dichloromethane (10 mL)was added dropwise. The solution was stirred overnight at roomtemperature. The mixture was diluted with dichloromethane (50 mL) andthe organic layer was washed sequentially with 1N NaHSO₄, aqueoussaturated NaHCO₃, and brine, and dried over sodium sulfate. The solventwas removed by evaporation under reduced pressure. Three compounds inthe mixture were shown on TLC. Since the impurities were less soluble inmethanol, repeated treatment with methanol followed by filtrationremoved most of the impurities. Column chromatography on silica gel (2%MeOH in CHCl₃) afforded the tripeptide 3 as amber glassy solid (156.1mg, 12%).

Preparation of L-Phe-L-Phe-homotaurine isobutyl ester

Concentrated HCl (0.7 mL) was added to a cold (0° C.) solution ofN-Boc-L-Phe-L-Phe-homotaurine isobutyl ester (202 mg, 0.343 mmol) inmethanol. The mixture was stirred at room temperature for 2 hours andthen left standing in the refrigerator overnight. The solvent wasremoved under reduced pressure and the residual solid was dried in vacuoto afford the L-Phe-L-Phe-homotaurine isobutyl ester as a white solid(171.8 mg, 95%).

Preparation of L-Phe-L-Phe-Homotaurine (Compound X)

A solution of N—BOC-L-phenylalanine-N-hydroxusuccinimide ester (400 mg,1.1 mmol) in a mixture of ethanol (6 mL) and 1,4-dioxane (4 mL) wasadded to a solution of L-Phe-Homotaurine (273 mg, 1.0 mmol) in 1N NaOH(1.05 mL), water (3 mL) and ethanol (4 mL). The mixture was stirred atroom temperature overnight. The solvent was removed under reducedpressure and the solid (601.9 mg) was suspended in a mixture of acetone(8 mL) and isopropanol (0.2 mL) and stirred overnight at roomtemperature. The mixture was heated under refluxed for 30 minutes andthen was cooled to room temperature. The white solid was collected byfiltration, washed with ether, then dried in the vacuum oven for 45minutes. The resulting solid (423.1 mg) was dissolved in a mixture ofwater/tert-butanol (7:3, 5 mL) and treated with Amberlite IR-120 plus(washed, 15 g dry weight) for 2 minutes at room temperature. The resinwas removed by filtration and washed 3 times with the mixed solvents ofwater and tert-butanol (10 mL). Concentrated HCl (4 mL) was added. Thesolvents were removed under reduced pressure, and the resulting solidwas dried in vacuum. The compound was purified by recrystallization froma mixed solvent of THF and MeOH. The resulting solid was heated underreflux in methanol (about 3 mL) to remove the yellowish color. The solidwas dried in vacuo. Compound X was obtained as white solid (84.4 mg,20%). The ¹H and ¹³C NMR were consistent with the structure.

Preparation of N-(3-aminopropane-1-sulfonyl)-phenylalanine, ethyl ester(Compound CL)

The 3-chloropropane-1-sulfonyl chloride (10 mmol, 1.21 mL) was addeddropwise to a cold (−10° C.) solution of L-phenylalanine ethyl ester (10mmol, 2.3 g) and 4-methylmorpholine (20 mmol, 2.2 mL) in dichloromethane(30 mL). The mixture was stirred for 30 minutes at −10° C. and for 2hours at room temperature. The mixture was diluted with dichloromethane(40 mL) and washed twice with water, once with brine, dried over sodiumsulfate. Evaporation of the solvent under reduced pressure gave rise toalmost pure 3-chloropropyl sulfonamide as a yellow-oil in quantitativeyield. The ¹H and ¹³C NMR were consistent with the structure.

A mixture of the 3-chloropropyl sulfonamide (10 mmol), sodium azide (20mmol) and a catalytic amount of Bu₄NI in DMF (40 ml) was heated at 60°C. for 24 hours. The mixture was diluted with ethyl acetate, washed withwater three times, and once with brine, dried over sodium sulfate.Evaporation of the solvent gave rise to the azide as a brown-oil (3.0489g, 8.96 mmol, 90%). The ¹H and ¹³C NMR were consistent with thestructure.

The azide (2.70 g, 7.94 mmol) was stirred under H₂ (40 psi) with 10%Pd/C (348 mg) in ethanol (16 mL) at room temperature overnight. Thesolid was removed by filtration over Celite. The filtrate was treatedwith TMSCl/EtOH in attempt to obtain a crystalline hydrochloride salt ofthe product. The solvent was evaporated to give a thick residue (2.2 g,6.27 mmol, 79%) that crystallized under none of the conditions tried.The crude hydrochloric acid salt was dissolved in dichloromethane andwashed once with saturated sodium bicarbonate. The organic layer wasrecovered and dried over sodium sulfate. The solvent was removed byevaporation under reduced pressure. The residue was dissolved inmethanol and treated with activated charcoal. The solid was removed byfiltration over Celite and the filtrate was evaporated to dryness. Theresidue was dried in vacuo to afford a tan oil (1.3969 g, 56% from theazide). The ¹H and ¹³C NMR were consistent with the structure ofCompound CL.

Preparation of N-Boc-L-Phe-(3-aminopropane-1-sulfonyl)-L-Phe, ethylester (Compound Y)

A solution of N-t-BOC-L-Phe N-hydroxysuccinimide ester (2.80 mmol, 1.01g) in dichloromethane (12 mL) was added to a cold (0° C.) solution of3-aminopropane-1-sulfonyl-L-Phe-OEt (2.66 mmol, 839 mg) indichloromethane (10 mL). The mixture was stirred overnight at roomtemperature. The mixture was diluted with dichloromethane, and washedwith 2N HCl, aqueous saturated NaHCO₃, and brine. The organic layer wasdried over magnesium sulfate and the solvent was removed under reducedpressure. The residue was applied on flash column chromatography onsilica gel (2% MeOH in CHCl₃). A portion of the pure desired material(600 mg) was isolated. The remaining product was mixed with 40% of thesuccinimide Some 3-aminopropanol (70 μL) was added to the mixture thatwas dissolved in dichloromethane (8 mL) and cooled to 0° C. The mixturewas stirred for 1 hour at room temperature. The mixture was diluted withdichloromethane, and washed with 2N HCl, aqueous saturated NaHCO₃,brine. The organic layer was dried over magnesium sulfate and thesolvent was removed under reduced pressure. The product was purified byflash column chromatography on silica gel (2% MeOH in CHCl₃). Anotherportion of the pure desired material (432.4 mg) was isolated along withthe adduct of the succinimide and 3-aminopropanol (220.6 mg, 0.684mmol). Compound Y was obtained as a white crystalline foamy solid (1030mg, 1.83 mmol, 65%). The ¹H and ¹³C NMR were consistent with thestructure.

Preparation of L-Phe-(3-aminopropane-1-sulfonyl)-L-Phe, ethyl ester(Compound Z)

Concentrated HCl (0.8 mL) was added to a cold (0° C.) solution of theN-Boc-L-Phe-(3-Aminopropane-1-Sulfonyl)-L-Phe, Ethyl Ester (197 mg,0.350 mmol) in ethanol (8 mL). The mixture was cooled using an ice/waterbath and stirred for 45 min and for 3 hours at room temperature. And themixture was kept in freezer (−20° C.) over the weekend. The solvent wasremoved under reduced pressure. The residual ethanol was removed bycoevaporation 3 times with chloroform under reduced pressure. Theresidue was dried in vacuo to give an off-white crystalline solid (175.3mg) in quantitative yield. The ¹H and ¹³C NMR were consistent with thestructure of Compound Z.

Preparation of L-(N-Boc)-Phe-(3-aminopropane-1-sulfonyl)-L-Phe, sodiumsalt (Compound AA)

One equivalent of 1N NaOH (202 μL) was added to a solution ofL-(N-Boc)-Phe-(3-aminopropane-1-sulfonyl)-L-Phe, ethyl ester (110 mg,0.197 mmol) in methanol (2 mL). The mixture was stirred overnight atroom temperature. A white suspension was observed after overnightstirring. MeOH (1 mL), water (1 mL) and 1N NaOH (10 μL) were added. Themixture was stirred in a warm water bath for about 2 hours. The solventmethanol was removed under reduced pressure. The wet residue was thenfreeze-dried to give compound AA as a white powder in quantitative yield(110.2 mg). The ¹H and ¹³C NMR were consistent with the structure.

Preparation of L-Phe-(3-aminopropane-1-sulfonyl)-L-Phe, methyl ester(Compound AB)

The hydrolysis was done by the conventional LiOH/MeOH method. Theproduct was purified by recrystallization from EtOAc and Hexanes.

The L-(N-Boc)-Phe-(3-aminopropane-1-sulfonyl)-L-Phe-OH (203 mg, 0.382mmol) was dissolved in methanol (4 mL) and the solution was cooled to 0°C. Concentrated HCl (0.35 mL) was added and the mixture was stirred for2 hours at 0° C. and for 2.5 h at room temperature. The volatilesolvents were removed under reduced pressure. The aqueous residue wasfreeze-dried to give the product as a white solid (171.4 mg). The NMRand MS showed to product to be a mixture of the free acid and the methylester. The MS showed also a strong association of the peptide. The dimerwas the major species on the MS. The solid was dissolved into methanoland treated with HCl overnight at room temperature. The solvent wasevaporated and the residue was dried in vacuo. The product was obtainedas a white foamy solid (180.4 mg, 97%). The ¹H and ¹³C NMR wereconsistent with the structure of compound AB.

Preparation of 4-iodo-N-(3-sulfopropyl)-L-phenylalanine amide (CompoundCO)

Thionyl chloride (8.2 mL, 112.5 mmol) was added to a cold MeOH (60 mL,in an ice-bath). The ice bath was removed and 4-iodo-L-phenylalanine(6.55 g, 22.4 mmol) was added to the mixture. The solution was stirredat reflux for 2 h. The solvent was removed under reduced pressure. Theresidual solid was dissolved in MeOH (40 mL) and the solution was pouredinto Et₂O (300 mL). The solid was collected by filtration, washed withEt₂O (2×50 mL) and dried in vacuo.

The solid (1.96 g, 5.8 mmol) was dissolved in a minimum amount of water.To the solution was added aqueous NH₄OH (28-30%, 15 mL). The reactionmixture was stirred at room temperature over weekend. The solvent wasremoved under reduced pressure and EtOAc (15 mL) was added. The mixturewas heated under reflux. The hot solution was filtered. The filtrate wascooled to room temperature and was stored in the fridge. The solid wascollected by filtration, washed with EtOAc, to give 4-iodophenylalanineamide. The amide (1.3 g, 4.4 mmol) was dissolved in 15 mL of 2-butanonewith a few drops of DMF before 1,3-propane sultone (560 mg, 4.9 mmol)was added. The reaction mixture was stirred at reflux for 2 hours. Themixture was cooled to room temperature. The solid was collected byfiltration, washed with acetone (2×20 mL) and dried in vacuo. The solidwas suspended in MeOH (25 mL) and a small amount of water (1 mL). Thesuspension was stirred at reflux. The solid material was collected byfiltration while the mixture was still hot. The solid was washed withhot MeOH (2×10 mL). Compound CO was obtained as a white solid (320 mg).

Preparation of3-[4-(4-fluorophenyl)-1,2,3,6-tetrahydropyridin-1-yl]-1-propanesulfonicacid (Compound F)

The 4-(4-fluorophenyl)-1,2,3,6-tetrahydropyridine hydrochloride (2.58 g,14.5 mmol) was treated with 1N NaOH (20 mL). The aqueous mixture wasextracted with CH₂Cl₂ (20 mL). The organic layer was separated and driedover MgSO₄. The solvents were removed by evaporation under reducedpressure.

To a solution of 4-(4-fluorophenyl)-1,2,3,6-tetrahydropyridine (1.96 g,13.7 mmol) in acetone (30 mL) was added 1,3-propane sultone (1.74 g,14.5 mmol). The mixture was stirred at reflux overnight. Only a smallamount of compound precipitated. The resulting suspension was cooled toroom temperature with stiffing and a larger amount of solidprecipitated. The suspension was heated with the addition of a smallamount of MeOH until complete dissolution of the solid. The resultingsolution was stirred at reflux for a few minutes and was cooled to roomtemperature with stiffing. The solid was collected by filtration, washedwith MeOH and dried in vacuo. This allowed the isolation of compound F,1.33 g (32%).

Preparation of3-[4-(4-bromophenyl)-4-hydroxypiperidin-1-yl]-1-propanesulfonic acid(Compound G)

To a solution of 4-(4-bromophenyl)-4-piperidinol (2.51 g, 9.8 mmol) inMeOH (25 mL) was added 1,3-propane sultone (1.28 g, 10.7 mmol). Themixture was stirred at reflux for 2 h. Only a small amount of compoundprecipitated. The resulting suspension was cooled to room temperaturewith stirring and a solution of 50% MeOH/Acetone was added toprecipitate a maximum of compound. The solid was collected byfiltration, washed with 50% MeOH/Acetone (2×25 mL) and dried in vacuo.This allowed the isolation of compound G, 2.11 g (57%).

Preparation of3-[4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl]-1-propanesulfonic acid(Compound H)

To a solution of 4-(4-chlorophenyl)-4-piperidinol (2.5 g, 11.8 mmol) inacetone (25 mL) was added 1,3-propane sultone (1.56 g, 13.0 mmol). Themixture was stirred at reflux for 2 h. The reaction mixture was cooledto room temperature. The solid was collected by filtration, washed withacetone (2×20 mL) and dried in vacuo. This allowed the isolation ofcompound H, 2.83 g (72%).

Preparation of 3-(4-acetyl-4-phenylpiperidin-1-yl)-1-propanesulfonicacid (Compound I)

4-Acetyl-4-phenylpiperidine hydrochloride (3.32 g, 12.5 mmol) wastreated with 1N NaOH (20 mL). The aqueous mixture was extracted withCH₂Cl₂ (20 mL). The organic layer was separated, dried over Na₂SO₄,filtered, and the solvent was removed under reduced pressure.

To a solution of 4-acetylphenylpiperidine (1.83 g, 9.0 mmol) in acetone(22 mL) was added 1,3-propane sultone (1.20 g, 10.0 mmol). The mixturewas stirred at reflux for 2 hours. The reaction mixture was cooled toroom temperature. The solid was collected by filtration, washed withacetone (2×20 mL) and dried in vacuo. This allowed the isolation ofcompound I, 2.65 g (90%).

Preparation of3-[4-(4-chlorophenyl)-1,2,3,6-tetrahydropyridin-1-yl]-1-propanesulfonicacid (Compound J)

The 4-(4-chlorophenyl)-1,2,3,6-tetrahydropyridine hydrochloride (2.52 g,10.9 mmol) was treated with 1N NaOH (20 mL); and aqueous mixture wasextracted with CH₂Cl₂ (20 mL). The organic layer was separated, driedover Na₂SO₄, and filtered. The solvent was removed under reducedpressure.

To a solution of 4-(4-chlorophenyl)-1,2,3,6-tetrahydropyridine (2.07 g,10.7 mmol) in acetone (25 mL) was added 1,3-propane sultone (1.41 g,11.8 mmol). The mixture was stirred at reflux for 2 h. The reactionmixture was cooled to room temperature. The solid was collected byfiltration, washed with acetone (2×20 mL) and dried in vacuo. Theproduct was suspended in 50% MeOH/acetone (75 mL). The suspension wasstirres at reflux for 5 minutes before 25 mL of cold acetone was added.The solid material was filtered and washed with acetone (2×25 mL). Thisallowed the isolation of compound J, 1.48 g (44%).

Preparation of 3-(4-phenylpiperazin-1-yl)-1-propanesulfonic acid(Compound K)

To a solution of 1-phenylpiperazine (2.0 g, 1.9 mL, 12.3 mmol) inacetone (20 mL) was added 1,3-propane sultone (1.53 g, 12.9 mmol). Themixture was stirred at reflux for 2 hours. The reaction mixture wascooled to room temperature. The solid was collected by filtration,washed with acetone (2×25 mL) and dried in vacuo. This allowed theisolation of compound K, 3.04 g (87%).

Preparation of 3-[4-(4-chlorophenyl)piperazin-1-yl]-1-propanesulfonicacid (Compound L)

The 1-(4-chlorophenyl)piperazine dihydrochloride (2.5 g, 9.3 mmol) wastreated with 1N NaOH (40 mL); and the aqueous mixture was extracted withCH₂Cl₂ (40 mL). The organic layer was separated, dried over Na₂SO₄,filtered and solvent was removed under reduced pressure.

To a solution of 1-(4-chlorophenyl)piperazine (1.62 g, 8.2 mmol) inacetone (20 mL) was added 1,3-propane sultone (1.06 g, 8.6 mmol). Themixture was stirred at reflux for 2 h. The reaction mixture was cooledto room temperature. The solid was collected by filtration, washed withacetone (2×25 mL) and dried in vacuo. This allowed the isolation ofcompound L, 2.11 g (81%).

Preparation of 3-[4-(2-fluorophenyl)piperazin-1-yl]-1-propanesulfonicacid (Compound M)

To a solution of 1-(2-fluorophenyl)piperazine (2.5 g, 2.2 mL, 13.9 mmol)in acetone (25 mL) was added 1,3-propane sultone (1.73 g, 14.6 mmol).The mixture was stirred at reflux for 2 hours. The reaction mixture wascooled to room temperature. The solid was collected by filtration,washed with acetone (2×25 mL) and dried in vacuo. This allowed theisolation of compound M, 3.56 g (85%).

Preparation of 3-[4-(4-nitrophenyl)piperazin-1-yl]-1-propanesulfonicacid (Compound N)

To a solution of 1-(4-nitrophenyl)piperazine (2.58 g, 12.1 mmol) inacetone (25 mL) was added 1,3-propane sultone (1.06 g, 8.6 mmol). Themixture was stirred at reflux for 2 h. The reaction was cooled to roomtemperature. The solid was collected by filtration, washed with acetone(2×25 mL) and dried in vacuo. This allowed the isolation of compound N,2.85 g (71%).

Preparation of 3-[4-(4-fluorophenyl)piperazin-1-yl]-1-propanesulfonicacid (Compound P)

To a solution of 1-(4-fluorophenyl)piperazine (2.0 g, 11.1 mmol) inacetone (20 mL) was added 1,3-propane sultone (1.46 g, 11.7 mmol). Themixture was stirred at reflux for 2 hours. The reaction mixture wascooled to room temperature. The solid was collected by filtration,washed with acetone (2×25 mL) and dried in vacuo. This allowed theisolation of compound P, 2.62 g (78%).

Preparation of 3-(4-phenyl-1,2,3,6-tetrahydropyridin-1-yl)propanoic acid(Compound Q)

The 4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (1.5 g, 7.8 mmol)suspended in 16 mL of CH₂Cl₂. To this suspension was added triethylamine(2.1 mL, 15.3 mmol) followed by methyl 3-bromopropionate (1.0 mL, 9.2mmol). The reaction was stirred at room temperature for 4 h and atreflux for 2 h. The reaction mixture was washed with water, 1N HCl (2×20mL), 1N NaOH (2×20 mL) and Brine (1×20 mL). The organic layer wasseparated, dried over Na₂SO₄, filtered; and solvent was removed underreduced pressure. To the crude product was added 2N NaOH (15 mL). Themixture was stirred at reflux for 1 h. The reaction mixture was washedwith CH₂Cl₂ (3×20 mL) and neutralized with concentrated HCl. The aqueoussolution was concentrated to dryness under reduced pressure, to give asolid residue. Sodium chloride in the residue was removed in thefollowing way (three repeats): dissolving the residue in a minimumamount of water, treating the aqueous solution with acetone, removingthe resultant solid material by filtration, and concentrate the filtrateto dryness under reduced pressure. This allowed the isolation ofcompound Q (159.4 mg).

Preparation of 3-dibenzylamino-1-propanesulfonic acid (Compound AV)

To a solution of dibenzylamine (9.8 mL, 50.8 mmol) in toluene (50 mL)was added 1,3-propane sultone (6.50 g, 53.3 mmol). The mixture wasstirred at reflux for 3 h. A sticky paste was formed at the bottom ofthe flask. The reaction mixture was cooled to room temperature. The toplayer was decanted; and the paste was partially dissolved in EtOAc withheating. The mixture was poured in a 10% EtOAc/Hexanes (200 mL). Themixture was heated and the paste was spread on the walls of the conicalflask. The solvent was removed. This process was repeated twice. MeOH(75 mL) was added to the paste. The mixture was heated until a whitesolid appeared. The solid was collected by filtration, washed with coldMeOH, and dried in vacuo, affording compound AV, 7.03 g (43%).

Preparation of 3-(4-cyano-4-phenylpiperidin-1-yl)-1-propanesulfonic acid(Compound E)

The 4-cyano-4-phenylpiperidine hydrochloride (2.0 g, 9.0 mmol) wastreated with 1N NaOH (20 mL) and the aqueous phase was extracted withCH₂Cl₂ (20 mL). The organic layer was separated, dried over MgSO₄,filtered and solvent was removed under reduced pressure.

To a solution of piperidine (1.43 g, 7.7 mmol) in acetone (20 mL) wasadded 1,3-propane sultone (1.02 g, 8.5 mmol). The mixture was stirred atreflux for 2 h. The resulting suspension was cooled to room temperature.The solid was collected by filtration, washed with acetone and dried invacuo. The solid was recrystallized from MeOH (and traces of water) toafford compound E, 800 mg (34%).

Preparation of 3-(4-phenyl-1,2,3,6-tetrahydropyridin-1-yl)butanoic acidhydrochloride

The 4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (2.01 g, 10.2mmol) was treated with 1N NaOH (20 mL) and the aqueous phase waextracted with CH₂Cl₂ (20 mL). The organic layer was separated, driedover MgSO₄, filtered and solvent was removed under reduced pressure.

The resulting 4-phenyl-1,2,3,6-tetrahydropyridine (1.55 g, 9.7 mmol) wasdissolved in 20 mL of 2-butanone. To this solution was added potassiumcarbonate (2.02 g, 14.6 mmol). The mixture was stirred 30 minutes atroom temperature; ethyl 4-bromobutyrate (1.46 mL, 10.1 mmol) was added.The reaction mixture was stirred at reflux for 5 hours. After cooling toroom temperature, inorganic salts were filtered. The solvent wasevaporated under reduced pressure. The residue was dissolved in CH₂Cl₂(30 mL). The organic phase was washed with water (2×30 mL), 2N HCl (2×30mL) and Brine (2×30 mL). The organic layer was dried with Na₂SO₄,filtered, evaporated under reduced pressure and dried in vacuo. Thisallowed the isolation of 1.55 g (58%) of the desired ester.

The ester (5.7 mmol) was dissolved in 6N HCl (40 mL). The reactionmixture was stirred at room temperature for 5 hours and at reflux for 1hour before it was cooled to room temperature. The reaction mixture wasextracted with CH₂Cl₂ (3×30 mL). The aqueous phase was evaporated underreduced pressure. The residue was dissolved in water (20 mL); and theaqueous solution was concentrated to dryness under reduced pressure. Theresultant material was further dried in vacuo, affording compound R, 973mg (61%).

Preparation of 3-piperonylamino-1-propanesulfonic acid (Compound AW)

To a solution of piperonylamine (2.5 mL, 19.8 mmol) in acetone (30 mL)was added 1,3-propane sultone (2.52 g, 20.8 mmol). The mixture stirredat reflux for 2 hours. The reaction mixture was cooled to roomtemperature. The solid material was collected by filtration, washed withacetone (2×25 mL) and dried in vacuo. The product was suspended in 90%Acetone/MeOH (75 mL). The suspension was stirred at reflux for 30 sec.,the solid was collected by filtration and dried in vacuo. This allowedthe isolation of compound AW, 2.56 g (45%).

Preparation of 3-(3,4,5-trimethoxybenzyl)amino-1-propanesulfonic acid(Compound AY)

To a solution of 3,4,5-trimethoxybenzylamine (2.2 mL, 12.7 mmol) in2-butanone (20 mL) was added 1,3-propane sultone (1.66 g, 13.3 mmol).The mixture was stirred at reflux for 2 hours. The reaction mixture wascooled to room temperature. The solid was collected by filtration,washed with acetone (2×25 mL) and dried in vacuo. The product wassuspended in 90% Acetone/MeOH (75 mL). The suspension was stirred atreflux for 30 sec., the solid was collected by filtration, and dried invacuo; affording compound AY, 2.61 g (64%).

Preparation of 3-(2,3-dimethoxybenzyl)amino-1-propanesulfonic acid(Compound AZ)

To a solution of 2,3-dimethoxybenzylamine (2.2 mL, 15.0 mmol) in2-butanone (20 mL) was added 1,3-propane sultone (1.97 g, 15.8 mmol).The mixture was stirred at reflux for 2 hours. The reaction mixture wascooled to room temperature. The solid was collected by filtration,washed with acetone (2×25 mL) and dried in vacuo. The crude product wassuspended in 90% Acetone/MeOH (75 mL). The suspension was stirred atreflux for 30 seconds, the solid was collected by filtration, and driedin vacuo; affording compound AZ, 1.95 g (45%).

Preparation of 3-(N-benzhydrylcarbamyl)amino-1-propanesulfonic acid(Compound AF)

The 3-amino-1-propanesulfonic acid (1.0 g, 7.2 mmol) was dissolved in 3NNaOH (370 mg, 9.4 mmol in 3 mL of water). After the solution was cooledto 0° C., diphenylmethyl isocyanate (1.4 mL, 7.2 mmol) was added. Thereaction mixture was allowed to warm up to room temperature, stirred for8 h (r.t.), and followed by addition of 3N NaOH (3 mL). The reactionmixture was stirred for 18 h. The pH of the reaction mixture was broughtto 3 with 5N HCl. The solvent was evaporated under reduced pressure.EtOH (15 mL) was added and the mixture was stirred at reflux for 30 sec.The hot mixture was filtered. The filtrate was evaporated to dryness.This process was repeated 2 more times. The final product was dried invacuo, affording compound AF, 837 mg (34%).

Preparation of 3-(3,5-dimethoxybenzyl)amino-1-propanesulfonic acid(Compound BA)

To a solution of 3,5-dimethoxybenzylamine (2.5 g, 15.0 mmol) in2-butanone (22 mL) was added 1,3-propane sultone (1.95 g, 15.8 mmol).The mixture was stirred at reflux for 2 hours. The reaction mixture wascooled to room temperature. The solid was collected by filtration,washed with acetone (2×25 mL), and dried in vacuo. This allowed theisolation of compound BA, 2.89 g (67%).

Preparation of 3-(2,4-dimethoxybenzyl)amino-1-propanesulfonic acid(Compound BB)

The 2,4-dimethoxybenzylamine hydrochloride (2.51 g, 12.3 mmol) wastreated with 1N NaOH (20 mL) and the aqueous phase was extracted withCH₂Cl₂ (20 mL). The organic layer was separated, dried over MgSO₄, andfiltered. Solvent was removed under reduced pressure to give the amine(free base form).

To a solution of 2,4-dimethoxybenzylamine (1.71 g, 10.3 mmol) in2-butanone (15 mL) was added 1,3-propane sultone (1.31 g, 10.7 mmol).The mixture was stirred at reflux for 2.5 h. The reaction mixture wascooled to room temperature. The supernatant was decanted; and the pastewas washed with acetone (2×30 mL), and dissolved in MeOH with heating.Acetone addition to the methanolic solution caused precipitation. Thesolid material was collected by filtration, washed with acetone (2×25mL), and dried in vacuo; affording compound BB, 1.14 g (38%).

Preparation of 3-(phenylacetamido)-1-propanesulfonic acid, sodium salt(Compound AG)

3-amino-1-propanesulfonic acid (1.0 g, 7.2 mmol) was dissolved insolution of 3M NaOH (7.2 mL). The mixture was cooled to 0° C. beforephenylacetyl chloride (1.4 mL, 10.8 mmol) was added. The reactionmixture was allowed to warm up to room temperature and it was stirredfor 22 h. The solvent was evaporated under reduced pressure. The residuewas suspended in 50% EtOH/Acetone. The mixture was stirred at reflux for30 sec. The solid material was collected by filtration and dried invacuo. The product was recrystallized from 95% EtOH/H₂O and dried invacuo. This allowed the isolation of compound AG, 880 mg (44%).

Preparation of 3-(N-benzylcarbamyl)amino-1-propanesulfonic acid, sodiumsalt (Compound AH)

3-amino-1-propanesulfonic acid (1.06 g, 7.7 mmol) was dissolved in 1.5NNaOH (5.3 mL). To this solution was added benzyl isocyanate (927 μL, 7.7mmol). The reaction mixture was stirred at 70° C. for 30 min, followedby addition to the mixture one-equivalent of benzyl isocyanate (927 μL,7.7 mmol). The reaction mixture was stirred for 1 hour. The solvent wasevaporated under reduced pressure. The residue was suspended in hotacetone. The solid material was collected by filtration, washed with hotacetone, and dried in vacuo; affording compound AH, 2.07 g (92%).

Preparation of 3-(N-n-dodecylcarbamyl)amino-1-propanesulfonic acid,sodium salt (Compound AJ)

3-amino-1-propanesulfonic acid (1.06 g, 7.7 mmol) was dissolved in 1.5NNaOH (5.3 mL). To this solution was added n-dodecyl isocyanate (1.7 mL,7.7 mmol). The reaction mixture was stirred at 70° C. for 30 minfollowed by addition to the mixture one-equivalent of n-dodecylisocyanate (1.7 mL, 7.7 mmol). The reaction mixture was stirred for 1 h.The solvent was removed under reduced pressure. The residual materialwas suspended in hot acetone. The solid material was collected byfiltration, washed with hot acetone, and dried in vacuo; affordingcompound AJ, 2.47 g (86%).

Preparation of 3-(N-1-adamantylcarbamyl)amino-1-propanesulfonic acid,sodium salt (Compound AK)

3-amino-1-propanesulfonic acid (1.06 g, 7.7 mmol) was dissolved in 1.5NNaOH (5.3 mL). To this solution was added 1-adamantyl isocyanate (1.36 g7.7 mmol) in hot EtOH (5 mL). The reaction mixture was stirred at 70° C.for 30 min followed by addition (to the mixture) of one-equivalent of1-adamantyl isocyanate (1.37, 7.7 mmol) in hot EtOH (5 mL). The reactionmixture was stirred for 1 h. The solvent was removed under reducedpressure. The residue was suspended in hot acetone. The solid materialwas collected by filtration, washed with hot acetone, and dried invacuo. The solid was recrystallized from EtOH, affording compound AK,519.4 mg (20%).

Preparation of 3-[2-(4-isobutylphenyl)propanoyl]amino-1-propanesulfonicacid, sodium salt (Compound AL)

Thionyl chloride (1.6 mL, 21.1 mmol) was added to ibuprofen (1.02 g, 4.9mmol). The reaction mixture was heated to reflux for 4 h. The solventwas evaporated, dried in vacuo, giving corresponding acid chloride.

3-amino-1-propanesulfonic acid (308 mg, 2.2 mmol) was dissolved in 1.5NNaOH (3 mL). To this solution was added dropwise the acid chloride(500.8 mg, 4.4 mmol, prepared above). The reaction mixture was stirredat 70° C. overnight. The solvent was removed under reduced pressure. Theresidue was suspended in acetone. The suspension was stirred at refluxfor 30 seconds. The solid material was removed by filtration. Thefiltrate was evaporated to dryness under reduced pressure. The residualmaterial was subjected to separation by flash chromatography (80%CH₂Cl₂/MeOH). This allowed the isolation of compound AL, 237 mg (14%).

Preparation of 3-[(benzylamino)thiocarbonyl]amino-1-propanesulfonicacid, sodium salt (Compound AM)

3-amino-1-propanesulfonic acid (1.07 g, 7.7 mmol) was dissolved in 1.5NNaOH (5.3 mL). To this solution was added benzyl isothiocyanate (1.02mL, 7.7 mmol). The reaction mixture was stirred at 70° C. for 0.5 h; asecond-equivalent of benzyl isocyanate (1.02 mL, 7.7 mmol) was added.The reaction mixture was stirred for 1 h. The solvent was evaporatedunder reduced pressure. The residue was suspended in hot acetone. Thesolid material was collected by filtration, washed with hot acetone, anddried in vacuo. The residual material was recrystallized from MeOH(traces of water), affording compound AM, 1.00 g (42%).

Preparation of 3-(3,4-dihydroxybenzyl)amino-1-propanesulfonic acid(Compound S)

To a solution of 3,4-dimethoxybenzylamine (2.2 mL, 15.0 mmol) in2-butanone (20 mL) was added 1,3-propane sultone (1.98 g, 15.8 mmol).The mixture was stirred at reflux for 2 hours. The reaction mixture wascooled to room temperature. The solid was collected by filtration,washed with acetone (2×25 mL) and dried in vacuo. The crude product wassuspended in 75% Acetone/MeOH (75 mL). The suspension was stirred atreflux for 30 sec.; the solid material was collected by filtration,washed with acetone (2×25 mL), and dried in vacuo. The solid (1.94 g,6.7 mmol) was dissolved hydrobromic acid (48%, 27 mL). The solution wasstirred at 100° C. for 4 h. The solvent was removed under reducedpressure. The residue was dissolved in water (20 mL). The aqueous phasewas washed with CH₂Cl₂ (3×20 mL), and evaporated under educed pressure.The solid residue was suspended in hot MeOH (75 mL). The suspension wasstirred at reflux for 30 sec.; the solid was collected by filtration,washed with 50% MeOH/acetone, and dried in vacuo. This allowed theisolation of compound S, 1.22 g (70%).

Preparation of 4-(3-phenylpropyl)-1-sulfopropylpyridinium hydroxide,inner salt (Compound C)

To a solution of 4-(3-phenylpropyl)pyridine (14.5 mL, 76 mmol) in2-butanone (150 mL) was added 1,3-propane sultone (10.0 g, 83.6 mmol).The mixture was stirred at reflux for 1.5 h. Once the reaction mixturewas cooled to room temperature, the precipitate was collected byfiltration and washed with acetone. The solid material wasrecrystallized from EtOH (traces of Et₂O), affording compound C, 15.8 g(66%).

Preparation of4-(3-phenylpropyl)-1-sulfopropyl-1,2,3,6-tetrahydropyridine (Compound D)

4-(3-phenylpropyl)-1-sulfopropylpyridine (10.7 g, 33.5 mmol) wasdissolved in 60 mL of MeOH. The solution was cooled to 0° C. beforesodium borohydride (2.55 g, 67.0 mmol) was added portionwise. Thereaction mixture was stirred for 0.5 hour at room temperature. Water (10mL) and concentrated HCl (5 mL) were successively added to the mixture.The inorganic material was removed by filtration. The filtrate wasconcentrated to dryness under reduced pressure and dried in vacuo. Theresultant viscous residue was dissolved in MeOH (60 mL). The solutionwas stirred with Amberlite IR-120 ion exchange resin (8.3 g) for 15 min.The resin was removed by filtration and washed with MeOH. The filtrateand washing was combined and concentrated to dryness under reducedpressure. The residual material was recrystallized from water, affordingcompound D (8.05 g, 75%) as white crystals.

Preparation of 3-ethylamino-1-propanesulfonic acid (Compound CV)

Tetrahydrofuran (THF, 800 mL) was placed a 3 neck 2-L flask (equippedwith a condenser) and cooled to 5° C. with an ice-bath. To the cold THFwas added aqueous ethylamine (70 wt. % solution in water, 85 mL, 1.07mol), followed by addition of a cold solution of 1,3-propane sultone(25.08 g, 201 mmol) in THF (100 mL) over 24-min period. The mixture wasstirred, while cooled with an ice-bath, for 1 h. The ice-bath wasremoved and the mixture was stirred at room temperature overnight. Itwas then heated under reflux for 1 h to distill off the ethylamine. Thehot mixture was biphasic. Upon cooling, a solid crystallized at thebottom of the flask. Ether (400 mL) was added and the mixture was cooledto −20° C. The supernatant was decanted. Methanol (about 120 mL) wasadded to the residue. The mixture was heated to reflux; and completedissolution of the solid material was achieved. After the solution wascooled to room temperature, precipitates formed. The mixture was cooledin an ice-bath; and the solid material was collected by filtration,rinsed with cold methanol and dried in vacuo (20.66 g, pure by NMRanalysis). The solid material was recrystallized from methanol (100 mL).After the mixture was cooled using an ice-bath, the solid was collectedby filtration, rinsed with cold methanol, and dried in a vacuum oven at40° C. Compound CV was obtained as white fine needles (19.12 g, 57%).The ¹H and ¹³C NMR were consistent with the structure.

Preparation of 3-(1-adamantyl)amino-1-propanesulfonic acid (Compound BW)

The 1-adamantanamine hydrochloride (80 g, 0.426 mol) was treated withNaOH (10%, 400 mL) in water. The aqueous mixture was extracted withdichloromethane (1×400 mL, 2×100 mL). The combined organic layers werewashed with brine (50 mL) and dried over sodium sulfate (10 g). Solventwas removed under reduced pressure. The resulting white waxy solid wasco-evaporated with acetonitrile (50 mL). The wet solid was suspended inacetonitrile (200 mL). The suspension mixture was added dropwise over 20min to a solution of 1,3-propane sultone (53 g, 0.426 mol) inacetonitrile (300 mL) and THF (200 mL). The thick mixture was stirredfor 2 hours under reflux with a mechanical stirrer. The suspension wasthen cooled to 13° C. The solid was collected by suction-filtration,rinsed with acetonitrile (2×100 mL) and ether (1×100 mL), air-dried for30 min, and further dried in vacuo at 60° C. overnight (104.17 g forcrop 1). Another crop was collected from the filtrate and dried in vacuoin the same manner (3.39 g for crop 2). Both crops gave identical protonNMR spectrum. The two batches were combined for further purification.The solid was suspended in methanol (720 mL) and the mixture was heatedto reflux. Water (490 mL) was added dropwise over 45 min, whilemaintaining reflux. After complete dissolution of the solid, thesolution was kept under reflux for 30 minute. The mixture was left inthe power-off heating mantle and allowed to cool slowly. After 90 min,the temperature reached 40° C. The heating mantle was replaced by athermostated water bath. The mixture was cooled to 5° C. and stirredovernight at this temperature. The white flaky solid was collected byfiltration, rinsed with cold (0° C.) methanol (2×125 mL), air-dried for60 minutes, and then dried in the vacuum oven at 60° C. overnight.Compound BW was obtained as a white flaky solid (white plates, 88.48 g,76% yield for the first crop). The ¹H NMR and MS were consistent withthe structure. A second crop (8.62 g) of compound BW was obtained fromthe mother liquid. The ¹H NMR was identical to that of the first crop.This made the reaction a total yield of 83% from the hydrochloride.

Preparation of 3-(2-norbornyl)amino-1-propanesulfonic acid (Compound BY)

To a solution of 2-aminonorbornane (7.3 g, 65.7 mmol) in 2-butanone (50mL) was added dropwise a solution of 1,3-propane sulfone (8.1 g, 65.7mmol) in 2-butanone (10 mL). The mixture was stirred at 60° C. for 1 h.The suspension was cooled to room temperature. The solid material wascollected by filtration and washed with ethanol (2×20 mL). The crudematerial was recrystallized from 95% EtOH to afford compound BY as awhite crystalline solid (8.2 g, 53% yield).

Preparation of 3-(2-adamantyl)amino-1-propanesulfonic acid (Compound BZ)

The 2-aminoadamantane hydrochloride (2×5 g) was treated with NaOH inwater. The aqueous mixture was extracted with dichloromethane. Theorganic layer was dried over magnesium sulfate. The solvent was removedunder reduced pressure. The resulting white solid was dried 30 minutesat room temperature under vacuum. A solution of 1,3-propane sultone (7.4g, 60 mmol) in THF was added to a solution of the free amine (7.98 g, 52mmol) in THF (70 mL, total). The mixture was heated under reflux for 4h, cooled into an ice bath. The solid was collected by filtration,air-dried for 15 min, and further dried in vacuo (11.2 g).Recrystallization was done with methanol/water (60 mL/35 mL). Aftercooled in a fridge, the solid was collected by filtration, rinsed withmethanol, dried in the vacuum oven at 60° C. overnight. A whitecrystalline sandy solid (small plates, 10.45 g, 74% yield) was obtained.The ¹H and ¹³C NMR were consistent with the structure of Compound BZ.

Preparation of 3-(3,4-dimethoxybenzyl)amino-1-propanesulfonic acid(Compound S)

To a solution of 3,4-dimethoxybenzylamine (2.2 mL, 15.0 mmol) in acetone(20 mL) was added 1,3-propane sultone (1.97 g, 15.8 mmol). The mixturewas stirred at reflux for 2 hours. The reaction mixture was cooled toroom temperature. The solid material was collected by filtration, washedwith acetone (2×25 mL) and dried in vacuo. The crude product wassuspended in 90% acetone/MeOH (75 mL). The suspension was stirred atreflux for 30 sec., the solid material was collected by filtration, anddried in vacuo. Compound S (1.84 g, 43%) was isolated as a white solid.¹H NMR (D₂O, 500 MHz) 8 ppm 6.96 (m, 3H), 4.06 (s, 2H), 3.74 (s, 6H),3.07 (t, 1H, J=7.8 Hz), 2.86 (t, 1H, J=7.8 Hz), 2.01 (m, 2H). ¹³C NMR(D₂O, 125 MHz) 8 ppm 149.23, 148.50, 123.63, 123.37, 55.90, 55.86,50.96, 48.06, 45.62, 21.39. ES-MS 290 (M+1).

Preparation of 3-(2,2-diphenylethyl)amino-1-propanesulfonic acid(Compound ET)

To a solution of 1,2-diphenylethylamine (2.49 g, 12.7 mmol) in2-butanone (15 mL) was added 1,3-propane sultone (1.67 g, 13.3 mmol).The mixture was stirred at reflux for 2 hours. The reaction mixture wascooled to room temperature. The solid material was collected byfiltration, washed with acetone (2×25 mL), and dried in vacuo, to givecompound ET: 3.02 g (74%). ¹H NMR (DMSO, 500 MHz) 8 ppm 8.58 (s (broad),1H), 7.34 (m, 8H), 7.23 (t, 2H, J=7.3 Hz), 4.32 (t, 1H, J=7.8 Hz), 3.68(d, 2H, J=7.3 Hz), 3.08 (t, 2H, J=6.1 Hz), 2.57 (t, 2H, J=7.3 Hz), 1.92(m, 2H). ¹³C NMR (DMSO, 125 MHz) 8 ppm 141.63, 129.46, 128.47, 127.76,50.85, 49.91, 48.53, 22.07. ES-MS 318 (M−1).

Preparation of 4-(tert-butylamino)-2-butanesulfonic acid (Compound ES)

To a solution of tert-butylamine (1.0 mL, 9.5 mmol) in tetrahydrofuran(15 mL) was added 2,4-butane sultone (1.33 g, 10.0 mmol). The mixturewas stirred at reflux for 2 hours. The reaction mixture was cooled toroom temperature. The solid product was collected by filtration, washedwith THF (2×20 mL), and dried in vacuo; affording compound ES: ¹H NMR(DMSO, 500 MHz) 8 ppm 2.97 (t, 2H, J=6.6 Hz), 2.62 (m, 1H), 1.95 (m,0.5H), 1.750 (m, 0.5H), 1.22 (s, 9H), 1.12 (d, 3H, J=6.8 Hz). ¹³C NMR(DMSO, 125 MHz) 8 ppm 56.17, 53.15, 29.87, 25.87, 17.05. ES-MS 207(M−1).

Preparation of 4-(tert-butylamino)-1-butanesulfonic acid (Compound ER)

To a solution of tert-butylamine (1.0 mL, 9.5 mmol) in tetrahydrofuran(4 mL) was added 1,4-butane sultone (1.36 g, 10.0 mmol) at roomtemperature. The solution was stirred at reflux for 2 hours. Thereaction mixture was cooled to room temperature. The solid product wascollected by filtration, washed with acetone (2×20 mL), and dried invacuo; affording compound ER 690 mg, (34%); ¹H NMR (D₂O, 500 MHz) 8 ppm2.92 (t, 2H, J=7.1 Hz), 2.82 (t, 2H, J=7.1 Hz), 1.68 (m, 4H), 1.22 (s,9H). ¹³C NMR (D₂O, 125 MHz) 8 ppm 57.07, 50.30, 40.95, 25.28, 24.96,21.62. ES-MS 210 (M−1).

Preparation of 3-(3-pentyl)amino-1-propanesulfonic acid (Compound DD)

To a solution of 1-ethylpropylamine (10.0 g, 115 mmol) intetrahydrofuran (80 mL) was added a solution of 1,3-propane sultone(13.7 g, 110 mmol) in 20 mL of THF. The solution was stirred at refluxfor 2 hours. The reaction mixture was cooled to room temperature. Thesolid product was collected by filtration, washed with acetone (2×50mL), and dried in vacuo., to afford compound DD (18.1, 80%): ¹H NMR(D₂O, 500 MHz) 8 ppm 3.08 (t, 2H, J=7.3 Hz), 3.01 (m, 1H), 2.87 (t, 2H,J=7.3 Hz) 2.00 (m, 2H), 1.59 (m, 4H), 0.82 (t, 6H, J=7.3 Hz). ¹³C NMR(D₂O, 125 MHz) 8 ppm 60.87, 48.14, 43.68, 21.81. 21.60, 8.25. ES-MS 208(M−1).

Preparation of 3-(tert-amyl)amino-1-propanesulfonic acid (Compound DG)

To a solution of tert-amylamine (2.0 g, 23.3 mmol) in tetrahydrofuran(15 mL) was added 1,3-propane sultone (2.76 g, 22.2 mmol). The solutionwas stirred at reflux for 2 hours. The reaction mixture was cooled toroom temperature. The solid product was collected by filtration, washedwith acetone (2×25 mL), and dried in vacuo, to afford compound DG (3.3g, 73%): ¹H NMR (D₂O, 500 MHz) 8 ppm 3.04 (t, 2H, J=7.8 Hz), 2.89 (t,2H, J=7.8 Hz), 2.87 (t, 2H, J=7.3 Hz), 1.97 (m, 2H), 1.55 (m, 2H), 1.18(s, 6H), 0.82 (t, 6H, J=7.3 Hz). ¹³C NMR (D₂O, 125 MHz) 8 ppm 60.42,48.17, 39.88, 30.81, 22.23, 21.98, 7.25. ES-MS 208 (M−1).

Preparation of 3-(1,1-dimethyl-2-hydroxyethyl)amino-1-propanesulfonicacid (Compound DH)

To a solution of 2-amino-2-methyl-1-propanol (2.0 g, 21.4 mmol) intetrahydrofuran (15 mL) was added 1,3-propane sultone (2.66 g, 21.4mmol). The mixture was stirred at reflux for 2 hours. The reactionmixture was cooled to room temperature. The crude product was collectedby filtration, washed with acetone (2×25 mL). The solid was suspended inEtOH (50 mL). The suspension was stirred at reflux for 5 minutes. Thesolid was collected by filtration and dried in a vacuum oven (50° C.),affording compound DH (2.5 g, 58%): ¹H NMR (D₂O, 500 MHz) 8 ppm 3.48 (s,2H), 3.04 (t, 2H, J=7.8 Hz), 2.90 (t, 2H, J=7.3 Hz), 2.00 (m, 2H), 1.18(s, 6H). ¹³C NMR (D₂O, 125 MHz) 8 ppm 64.88, 60.27, 48.19, 40.10, 21.92,20.02. ES-MS 210 (M−1).

Preparation of 3-(1-carboxy-1-methylethylamino)-1-propanesulfonic acid(Compound DI)

To a cold (5° C.) mixture of 2-aminoisobutyric acid (2.0 g, 19.4 mmol),NaOH (776 mg, 19.4 mmol) in 1,4-dioxane (10 mL) and water (4 mL) wasadded via syringe pump (over a 4-hour period), a solution 1,3-propanesultone (2.02 g, 16.2 mmol) in 1,4-dioxane (total: 4 mL). The solutionwas stirred at room temperature for 2 hours before it was allowed towarm up to room temperature. The reaction mixture was stirred underthese conditions overnight. The solvent was evaporated under reducedpressure. The resultant solid material was recrystallized from 5%water/EtOH. The resulting solid was dissolved in water; and the aqueoussolution was passed through an ion exchange column (Dowex 50WX 8, 100 g,solvent: water). The solvent was evaporated under reduced pressure. Theproduct was lyophilized to afford Compound DI (880 mg, 28%). ¹H NMR(D₂O, 500 MHz) 8 ppm 3.00 (t, 2H, J=7.6 Hz), 2.91 (t, 2H, J=7.3 Hz),2.01 (m, 2H), 1.34 (s, 6H). ¹³C NMR (D₂O, 125 MHz) 8 ppm 176.93, 63.89,48.13, 42.04, 22.15, 21.86. ES-MS 224 (M−1).

Preparation of 3-[(1R,2S)-2-methylcyclohexyl]amino-1-propanesulfonicacid (Compound DJ)

To a solution of 2-methylcyclohexylamine (98% cis and trans isomers,10.0 g, 88.3 mmol) in tetrahydrofuran (60 mL) was slowly added asolution of 1,3-propane sultone (10.5 g, 84.1 mmol) in THF (20 mL). Themixture was stirred at reflux for 2 hours. The reaction mixture wascooled to room temperature. The solid material was collected byfiltration, washed with acetone (2×50 mL). The solid was dissolved in50% EtOH/water (200 mL); and solution was treated with Dowex 50WX8 resin(15 g). The suspension stirred at room temperature for 15 min. The resinwas removed by filtration. The filtrate was concentrated to half of theoriginal volume on a rotary evaporator. The solid product slowlycrystallized. The product was collected by filtration, washed withacetone (2×50 mL) and dried in a vacuum oven (50° C.), to affordcompound DJ (10.4 g, 53%): ¹H NMR (D₂O, 500 MHz) δ ppm 3.15 (m, 1H),3.03 (m, 1H), 2.88 (t, 2H, J=7.3 Hz), 2.76 (m, 1H), 1.98 (m, 3H), 1.68(m, 2H), 1.51 (m, 2H), 1.18 (m, 3H), 1.01 (m, 1H), 0.92 (m, 3H). ¹³C NMR(D₂O, 125 MHz) δ ppm 62.97, 48.16, 42.72, 34.77, 33.50, 27.57, 24.51,24.23, 21.51, 17.80. ES-MS 234 (M−1).

Preparation of 3-(2,3-dimethylcyclohexyl)amino-1-propanesulfonic acid(Compound DK)

To a solution of 2,3-dimethylcyclohexylamine (10.0 g, 79.0 mmol) intetrahydrofuran (60 mL) was slowly added a solution of 1,3-propanesultone (9.3 g, 75.0 mmol) in THF (20 mL). The solution was stirred atreflux for 2 hours. The reaction mixture was cooled to room temperature.The solid was collected by filtration, washed with THF (50 mL) andacetone (50 mL). The solid was dissolved in 25% EtOH/water (150 mL), andtreated with Dowex 50WX8 resin (15 g). The suspension stirred at roomtemperature for 5 minutes. The resin was removed by filtration. Thefiltrate was concentrated to dryness under reduced pressure; and thesolid residue was suspended in acetone (100 mL). The solid material wascollected by filtration, and dried in vacuo; affording compound DK (7.4g, 43%). ¹H NMR (D₂O, 500 MHz) 8 ppm 3.29 (m, 0.5H), 3.09 (m, 2H), 2.88(t, 2H, J=7.3 Hz), 2.80 (m, 0.5H), 1.99 (m, 3H), 1.40 (m, 7H), 0.81 (m,6H). ¹³C NMR (D₂O, 125 MHz) 8 ppm 62.85, 61.56, 59.88, 58.22, 48.26,48.15, 44.29, 43.84, 43.59, 42.65, 42.01, 41.03, 37.34, 36.12, 34.73,34.45, 33.88, 33.64, 29.39, 28.00, 26.50, 24.25, 24.02, 23.64, 22.42,21.55, 21.45, 21.35, 19.38, 19.13, 18.82, 18.40, 14.38, 13.39, 4.59.ES-MS 248 (M−1).

Preparation of 3-neopentylamino-1-propanesulfonic acid (Compound DL)

To a solution of neopentylamine (8.5 g, 98 mmol) in tetrahydrofuran (75mL) was slowly added a solution of 1,3-propane sultone (11.5 g, 93 mmol)in THF (20 mL). The solution was stirred at reflux for 2 hours. Thereaction mixture was cooled to room temperature. The solid was collectedby filtration, washed with acetone (2×50 mL). The solid was suspended inEtOH (150 mL). The suspension was stirred at reflux for 15 minutes. Thesolid product was collected by filtration, washed with acetone (2×50mL), and dried in vacuo, to afford compound DL (13.3 g, 69%). ¹H NMR(D₂O, 500 MHz) 8 ppm 3.09 (t, 2H, J=7.3 Hz), 2.89 (t, 2H, J=7.3 Hz),2.78 (s, 2H), 2.04 (m, 2H), 0.901 (s, 9H). ¹³C NMR (D₂O, 125 MHz) 8 ppm59.44, 48.31, 47.83, 29.91, 26.42, 21.06. ES-MS 208 (M−1).

Preparation of 3-cumylamino-1-propanesulfonic acid (Compound DM)

To a solution of cumylamine (10.5 g, 78 mmol) in tetrahydrofuran (75 mL)was slowly added a solution of 1,3-propane sultone (9.2 g, 74 mmol) inTHF (20 mL). The mixture was stirred at reflux for 4 h. The reactionmixture was cooled to room temperature. The solid was collected byfiltration, and washed with THF (2×35 mL). The solid was suspended inEtOH (80 mL). The suspension was stirred at reflux for 15 minutes. Thesolid product was collected by filtration, washed with EtOH (35 mL) andacetone (35 mL). The resulting solid was dried in vacuo, affordingcompound DM (5.6 g, 30%). ¹H NMR (D₂O, 500 MHz) 8 ppm 7.41 (m, 5H), 2.74(m, 4H), 1.88 (m, 2H), 1.66 (s, 6H). ¹³C NMR (D₂O, 125 MHz) 8 ppm138.36, 129.44, 129.41, 126.52, 61.56, 48.04, 41.21, 24.80, 21.81. ES-MS256 (M−1).

Preparation of 3-[(1R)-1-(4-methylphenyl)ethyl]amino-1-propanesulfonicacid (Compound FN)

To a solution of (R)-(+)-1-(4-methoxyphenyl)ethylamine (5.83 g, 38.6mmol) in tetrahydrofuran (25 mL) was slowly added 1,3-propanesultone(4.56 g, 36.8 mmol). The solution was stirred at reflux for 4 hours. Thereaction mixture was cooled to room temperature. The solid was collectedby filtration, washed with THF (25 mL) and acetone (25 mL). The solidwas suspended in EtOH (200 mL). The suspension was stirred at reflux for15 min. The solid was collected by filtration, washed with cold EtOH (50mL), and dried in a vacuum oven (50° C.), to afford compound FN (5.6 g,56%). ¹H NMR (D₂O, 500 MHz) δ ppm 7.26 (d, 2H, J=8.3 Hz), 6.90 (d, 2H,J=8.3 Hz), 4.22 (m, 1H), 3.68 (s, 3H), 2.92 (m, 1H), 2.76 (m, 3H), 1.90(m, 2H), 1.49 (d, 3H, J=6.8 Hz). ¹³C NMR (D₂O, 125 MHz) 8 ppm 159.87,129.34, 128.18, 114.85, 57.99, 55.58, 48.05, 44.21, 21.45, 18.21. ES-MS272 (M−1).

Preparation of 3-[(1R)-1-indanamino]-1-propanesulfonic acid (CompoundDO)

To a solution of (R)-(−)-1-aminoindan (1.0 g, 7.5 mmol) intetrahydrofuran (10 mL) was slowly added 1,3-propane sultone (890 mg,7.1 mmol). The mixture was stirred at reflux for 2 hours. The reactionmixture was cooled to room temperature. The solid material was collectedby filtration, and washed with THF (20 mL) and acetone (20 mL). Thesolid was suspended in 80% acetone/EtOH (40 mL). The suspension wasstirred at reflux for 30 sec. The solid product was collected byfiltration, washed with acetone (2×20 mL), and dried in vacuo, to affordcompound DO (1.1 g, 61%). ¹H NMR (D₂O, 500 MHz) 8 ppm 7.41 (d, 1H, J=7.3Hz), 7.30 (m, 2H), 7.24 (m, 1H), 4.72 (m, 1H), 3.14 (t, 2H, J=7.8 Hz),3.02 (m, 1H), 2.88 (m, 3H), 2.43 (m, 1H), 2.12 (m, 1H), 2.01 (m, 2H).¹³C NMR (D₂O, 125 MHz) δ ppm 145.38, 136.39, 130.31, 127.20, 125.72,125.54, 62.98, 48.10, 43.93, 29.84, 28.62, 21.64. [α]_(D)=−1.3°(c=0.00515 in water). ES-MS 254 (M−1).

Preparation of 3-(N-tert-butylcarbamyl)amino-1-propanesulfonic acid,sodium salt (Sodium Salt of Compound DP)

3-amino-1-propanesulfonic acid (2.0 g, 14.3 mmol) was dissolved in 1.6MNaOH (10 mL). To this solution was added tert-butyl isocyanate (1.1 g,14.3 mmol). The reaction mixture was stirred at 70° C. for 1 h, followedby addition of one equivalent of tert-butyl isocyanate (1.1 g, 14.3mmol). The reaction mixture was stirred for 1 h. The solvent wasevaporated under reduced pressure. The residue was suspended in EtOH (30mL). The solid product was collected by filtration, washed with EtOH (20mL) and acetone (20 mL). The resulting solid was dried in vacuo, toafford the sodium salt of compound DP (2.1 g, 66%). ¹H NMR (D₂O, 500MHz) δ ppm 3.03 (t, 2H, J=6.6 Hz), 2.76 (t, 2H, J=7.6 Hz), 1.72 (m, 2H),1.12 (s, 9H). ¹³C NMR (D₂O, 125 MHz) δ ppm 160.35, 50.26, 48.74, 38.31,28.86, 25.24. ES-MS 280 (M+Na).

Preparation of 3-(1,2-dimethyl-1-propyl)amino-1-propanesulfonic acid(Compound DQ)

To a solution of 1,2-dimethylpropylamine (10.0 g, 115 mmol) intetrahydrofuran (80 mL) was slowly added a solution of 1,3-propanesultone (13.7 g, 110 mmol) in THF (20 mL). The solution was stirred atreflux for 2 hours. The reaction mixture was cooled to room temperature.The solid product was collected by filtration, washed with THF (50 mL)and EtOH (50 mL). The resulting solid was dried in vacuo, to affordcompound DQ (17.5 g, 76%). ¹H NMR (D₂O, 500 MHz) δ ppm 3.07 (m, 3H),2.88 (t, 2H, J=7.3 Hz), 1.97 (m, 3H), 1.10 (d, 3H, J=6.8 Hz), 0.85 (d,3H, J=6.8 Hz), 0.81 (d, 3H, J=6.8 Hz). ¹³C NMR (D₂O, 125 MHz) δ ppm59.79, 48.19, 44.18, 29.72, 21.51, 18.44, 15.02, 10.71. ES-MS 232(M+Na).

Preparation of 3-(4-methylcyclohexyl)amino-1-propanesulfonic acid(Compound DR)

To a solution of 4-methylcyclohexylamine (97% cis and trans isomers,11.0 g, 97.4 mmol) in tetrahydrofuran (70 mL) was slowly added asolution of 1,3-propane sultone (11.5 g, 92.8 mmol) in THF (20 mL). Themixture was stirred at reflux for 2 hours. The reaction mixture wascooled to room temperature. The solid material was collected byfiltration, washed with THF (50 mL) and acetone (50 mL). The solid wassuspended EtOH. The suspension was stirred at room temperature for 5minutes. The solid product was collected by filtration, washed with EtOH(50 mL), and dried in a vacuum oven (50° C.), to afford compound DR(16.1 g, 75%). ¹H NMR (D₂O, 500 MHz) δ ppm 3.08 (m, 2.5H), 2.93 (m,0.5H), 2.87 (m, 2H), 1.99 (m, 4H), 1.64 (m, 3H), 1.47 (m, 1H), 1.24 (m,2H), 0.88 (m, 1H), 0.78 (m, 3H). ¹³C NMR (D₂O, 125 MHz) δ ppm 57.35,56.52, 48.16, 18.05, 43.73, 43.30, 32.45, 31.13, 28.92, 28.69, 27.77,24.55, 21.65, 21.53, 21.20, 18.38. ES-MS 236 (M+1).

Preparation of 3-(2-methyl-1-butyl)amino-1-propanesulfonic acid(Compound DS)

To a solution of (+/−)-2-methylbutylamine (10 g, 115 mmol) intetrahydrofuran (80 mL) was slowly added a solution of 1,3-propanesultone (13.5 g, 109 mmol) in THF (20 mL). The mixture was stirred atreflux for 2 hours. The reaction mixture was cooled to room temperature.The solid material was collected by filtration, washed with acetone(2×30 mL). The solid was suspended 95% Acetone/EtOH (200 mL). Thesuspension was stirred at room temperature for 5 minutes. The solidproduct was collected by filtration, and dried in a vacuum oven (50°C.), to afford compound DS (17.6 g, 78%). ¹H NMR (D₂O, 500 MHz) δ ppm3.07 (t, 2H, J=7.8 Hz), 2.93 (m, 3H), 2.76 (m, 1H), 2.01 (m, 2H), 1.67(m, 1H), 1.30 (m, 1H), 1.09 (m, 1H), 0.81 (d, 3H, J=6.8 Hz), 0.77 (t,3H, J=6.8 Hz). ¹³C NMR (D₂O, 125 MHz) δ ppm 53.48, 48.13, 46.92, 31.96,26.38, 21.29, 16.11, 10.19. ES-MS 210 (M+1).

Preparation of 3-pivaloylamino-1-propanesulfonic acid (Compound DT)

3-amino-1-propanesulfonic acid (2.0 g, 14.4 mmol) was dissolved a NaOH(1.2 g, 30.2 mmol) solution in a mixture of 1,4-dioxane (5 mL) and water(15 mL). The mixture was cooled to 0° C. before pivaloyl chloride (2.8mL, 21.6 mmol) in 1,4-dioxane (5 mL) was added dropwise. The reactionmixture was allowed to warm up to room temperature and it was stirred at65° C. for 4 h. The solvent was evaporated under reduced pressure. Theresulting solid was dissolved in water (30 mL), and treated with Dowex50WX8 resin. The suspension was stirred for 5 minutes and the resin wasremoved by filtration. The filtrate was evaporated under reducedpressure. The residual material was suspended in 20% EtOH/Acetone. Themixture was stirres at reflux for 30 seconds. The solid product wascollected by filtration, and dried in vacuo, to afford compound DT (1.3g, 41%). ¹H NMR (D₂O, 500 MHz) δ ppm 3.16 (t, 2H, J=6.8 Hz), 2.75 (t,2H, J=7.8 Hz), 1.78 (m, 2H), 1.1 (s, 9H). ¹³C NMR (D₂O, 125 MHz) δ ppm182.75, 48.70, 38.57, 38.18, 26.65, 24.27. ES-MS 222 (M−1).

Preparation of 3-(3,3,5-trimethylcyclohexyl)amino-1-1-propanesulfonicacid (Compound ED)

To a solution of 3,3,5-trimethylcyclohexylamine (5.0 g, 35.4 mmol) intetrahydrofuran (35 mL) was slowly added a solution of 1,3-propanesultone (4.17 g, 33.7 mmol) in THF. The mixture was stirred at refluxfor 2 hours. The reaction mixture was cooled to room temperature. Thesolid material was collected by filtration, washed with acetone (2×25mL). The solid was suspended 90% Acetone/EtOH (100 mL) The suspensionwas stirred at room temperature for 5 min. The solid product wascollected by filtration, and dried in a vacuum oven (50° C.), affordingcompound ED (5.9 g, 67%). ¹H NMR (D₂O, 500 MHz) δ ppm 3.20 (m, 1H), 3.08(m, 2H), 2.87 (t, 2H, J=6.8 Hz), 1.96 (m, 3H), 1.60 (m, 2H), 1.29 (m,1H), 1.01 (m, 1H), 0.84 (s, 3H), 0.73 (m, 8H). ¹³C NMR (D₂O, 125 MHz) δppm 54.98, 48.06, 46.51, 43.28, 40.96, 37.02, 32.07, 31.23, 26.68,24.28, 21.67, 21.52. ES-MS 262 (M−1).

Preparation of 3-(2-indanamino)-1-propanesulfonic acid (Compound EE)

To a solution of 2-aminoindan (2.50 g, 18.8 mmol) in tetrahydrofuran (25mL) was slowly added 1,3-propane sultone (2.24 g, 17.9 mmol). Themixture was stirred at reflux for 2.5 h. The reaction mixture was cooledto room temperature. The solid material was collected by filtration,washed with acetone (2×25 mL). The crude product was suspended in 90%Acetone/EtOH (100 mL). The suspension was stirred at room temperaturefor 5 minutes. The solid product was collected by filtration, and driedin a vacuum oven (50° C.), to afford compound EE (3.1 g, 67%). ¹H NMR(DMSO, 500 MHz) δ ppm 7.25 (m, 2H), 7.20 (m, 2H), 4.00 (m, 1H), 3.30 (m,2H), 3.13 (m, 2H), 3.02 (m, 2H), 2.64 (m, 2H), 1.96 (m, 2H). ¹³C NMR(DMSO, 125 MHz) δ ppm 130.98, 127.80, 125.25, 57.70, 49.24, 45.87,36.32, 22.66. ES-MS 254 (M−1).

Preparation of 3-(4-biphenylamino)-1-propanesulfonic acid (Compound EF)

To a solution of 4-aminobiphenyl (3.0 g, 17.8 mmol) in tetrahydrofuran(25 mL) was slowly added 1,3-propane sultone (2.11 g, 16.9 mmol). Thesolution was stirred at reflux for 3 h. The reaction mixture was cooledto room temperature. The solid material was collected by filtration,washed with acetone (2×25 mL). The crude product was dissolved in a hotsolution of 80% MeOH/H₂O (120 mL). To this warm solution was added Dowex50WX8 ion exchange resin (10 g). The hot suspension was stirred for 5minutes and the resin was removed by filtration. The filtrate wasconcentrated to dryness under reduced pressure. The residual solid wasdried in a vacuum oven (50° C.), affording compound EF (283 mg, 6%). ¹HNMR (DMSO, 500 MHz) δ ppm 7.77 (d, 2H, J=7.8 Hz), 7.66 (d, 2H, J=7.8Hz), 7.46 (m, 3H), 7.37 (m, 1H), 3.44 (m, 2H), 2.68 (m, 2H), 1.98 (m,2H). ¹³C NMR (DMSO, 125 MHz) δ ppm 139.74, 129.69, 128.74, 128.33,127.31, 122.23, 49.70, 22.93. ES-MS 290 (M−1).

Preparation of3-[(1R,2S)-2-hydroxy-1-(methoxymethyl)-2-phenylethyl]amino-1-propanesulfonicacid (Compound EG)

To a solution of (1S,2S)-2-amino-3-methoxy-1-phenyl-1-propanol (1.0 g,5.5 mmol) in tetrahydrofuran (10 mL) was slowly added 1,3-propanesultone (662 mg, 5.3 mmol). The mixture was stirred at reflux for 2.5 h.The reaction mixture was cooled to room temperature. The solid materialwas collected by filtration, washed with acetone (2×25 mL). The crudeproduct was suspended 80% Acetone/EtOH. The suspension was stirred atreflux for 30 seconds. The solid product was collected by filtration anddried in a vacuum oven (50° C.), to afford compound EG (1.0 g, 63%). ¹HNMR (D₂O, 500 MHz) δ ppm 7.32 (m, 5H), 4.77 (d, 1H, J=9.8 Hz), 3.53 (m,1H), 3.37 (m, 1H). 3.26 (m, 1H), 3.17 (m, 6H), 2.91 (t, 2H, J=7.3 Hz),2.07 (m, 2H). ¹³C NMR (D₂O, 125 MHz) δ ppm 139.09, 129.33, 129.27,127.11, 70.85, 66.29, 62.62, 58.76, 48.14, 44.24, 21.47. [α]_(D)=+42.6°(c=0.00091 in water), ES-MS 302 (M−1).

Preparation of3-[(1R,2R,3R,5S)-1,2,6,6-tetramethylbicyclo[3.1.1]hept-3-yl]amino-1-propanesulfonicacid (Compound EH)

To a solution of (1R,2R,3R,5S)-(−)-isopinocampheylamine (2.0 g, 13.0mmol) in tetrahydrofuran (20 mL) was slowly added 1,3-propanesultone(1.56 g, 12.5 mmol). The mixture was stirred at reflux for 2 hours. Thereaction mixture was cooled to room temperature. The solid product wascollected by filtration, washed with acetone (2×25 mL), and dried in avacuum oven (50° C.), to afford compound EH (2.7 g, 80%). ¹H NMR (DMSO,500 MHz) δ ppm 3.32 (m, 2H), 3.09 (d, 2H), 2.67 (m, 2H), 2.30 (m, 2H),1.96 (m, 4H), 1.75 (m, 2H), 1.18 (s, 3H), 1.11 (m, 4H), 0.90 (s, 3H).¹³C NMR (DMSO, 125 MHz) δ ppm 55.97, 50.11, 47.55, 45.86, 41.15, 40.91,38.94, 32.81, 31.61, 27.96, 23.85, 22.59, 21.21, [α]_(D)=−17.2°(c=0.00083 in water), ES-MS 274 (M−1).

Preparation of 3-(2-methoxy-1-methylethyl)amino-1-propanesulfonic acid(Compound EI)

To a solution of 2-amino-1-methoxypropane (5.0 g, 17.8 mmol) intetrahydrofuran (25 mL) was slowly added 1,3-propane sultone (2.12 g,17.0 mmol). The mixture was stirred at reflux for 4 h. The reactionmixture was cooled to room temperature. The solid product was collectedby filtration, washed with acetone (2×25 mL), and dried in a vacuum oven(50° C.), to afford compound EI (3.1 g, 86%). ¹H NMR (D₂O, 500 MHz) δppm 3.53 (m, 1H), 3.41 (m, 2H), 3.27 (s, 3H), 3.11 (m, 2H), 2.89 (t, 2H,J=7.3 Hz), 2.00 (m, 2H), 1.18 (d, 3H, J=5.9 Hz). ¹³C NMR (D₂O, 125 MHz)δ ppm 71.73, 58.80, 53.79, 48.08, 43.55, 21.52, 12.79. ES-MS 210 (M−1).

Preparation of 3-[(1R)-2-benzyl-1-hydroxyethyl]amino-1-propanesulfonicacid (Compound EJ)

To a solution of (R)-(+)-2-amino-3-phenyl-1-propanol (1.0 g, 6.6 mmol)in tetrahydrofuran (10 mL) was slowly added 1,3-propane sultone (785 mg,6.3 mmol). The mixture was stirred at reflux for 2 hours. The reactionmixture was cooled to room temperature. The solid material was collectedby filtration, washed with acetone (2×25 mL). The crude product wassuspended 80% acetone/EtOH (100 mL). The suspension was stirred atreflux for 30 seconds. The solid product was collected by filtration,washed with acetone (2×25 mL), and dried in a vacuum oven (50° C.), toafford compound EJ (890 mg, 52%). ¹H NMR (DMSO, 500 MHz) δ ppm 8.58 (s(broad), 1H), 7.26 (m, 5H), 5.30 (s (broad), 1H), 3.53 (m, 1H), 3.31 (m,2H). 3.14 (t, 2H), 2.98 (m, 1H), 2.80 (m, 1H), 2.62 (t, 2H), 1.98 (m,2H). ¹³C NMR (DMSO, 125 MHz) δ ppm 137.31, 130.00, 129.26, 127.49,60.16, 57.78, 49.90, 45.34, 33.78, 22.49. [α]_(D)=+9.7° (c=0.00118 inwater). ES-MS 272 (M−1).

Preparation of 3-[1(1S)-2-benzyl-1-hydroxyethyl]amino-1-propanesulfonicacid (Compound EK)

To a solution of (S)-(−)-2-amino-3-phenyl-1-propanol (2.0 g, 13.2 mmol)in tetrahydrofuran (20 mL) was slowly added 1,3-propane sultone (1.57,12.6 mmol). The mixture was stirred at reflux for 2 hours. The reactionmixture was cooled to room temperature. The solid material was collectedby filtration, washed with acetone (2×25 mL). The crude product wassuspended 80% Acetone/EtOH. The suspension was stirred at reflux for 30seconds. The solid product was collected by filtration, and dried in avacuum oven (50° C.), to afford compound EK (1.9 g, 56%). ¹H NMR (DMSO,500 MHz) δ ppm 8.63 (s (broad), 1H), 7.27 (m, 5H), 5.31 (s (broad), 1H),3.53 (m, 1H), 3.25 (m, 2H). 3.15 (t, 2H), 2.98 (m, 1H), 2.80 (m, 1H),2.61 (t, 2H), 1.99 (m, 2H). ¹³C NMR (DMSO, 125 MHz) δ ppm 137.31,130.01, 129.27, 127.49, 60.18, 57.77, 49.88, 45.32, 33.78, 22.49.[α]_(D)=−7.5° (c=0.00118, H₂O). ES-MS 272 (M−1).

Preparation of 3-(N-methyl-N-tert-butylamino)-1-propanesulfonic acid(Compound EN)

To a solution of N-methyl-tert-butylamine (2.0 g, 22.9 mmol) in acetone(25 mL) was slowly added 1,3-propane sultone (2.72 g, 21.8 mmol). Themixture was stirred at reflux for 3 h. The reaction mixture was cooledto room temperature. The solid material was collected by filtration,washed with acetone (2×25 mL). The crude product was suspended in 80%Acetone/EtOH. The suspension was stirred at reflux for 30 seconds. Thesolid product was collected by filtration, and dried in a vacuum oven(50° C.), to afford compound EN (2.9 g, 65%). ¹H NMR (DMSO, 500 MHz) δppm 9.40 (s (broad), 1H), 3.45 (m, 1H), 2.85 (m, 1H), 2.59 (m, 5H), 1.99(m, 2H), 1.28 (s, 9H). ¹³C NMR (DMSO, 125 MHz) δ ppm 63.23, 51.12,49.73, 34.79, 25.50, 21.72. ES-MS 208 (M−1).

Preparation of 3-[(1R,2S)-2-hydroxyindan-1-amino]-1-propanesulfonic acid(Compound EO)

To a solution of (1R,2S)-1-amino-2-indanol (2.37 g, 15.9 mmol) intetrahydrofuran (25 mL) was slowly added 1,3-propane sultone (1.89 g,15.1 mmol). The mixture was stirred at reflux for 2 hours. The reactionmixture was cooled to room temperature. The solid material was collectedby filtration, and washed with acetone (2×25 mL). The crude product wassuspended in 80% acetone/ethanol (75 mL). The suspension was stirred atreflux for 30 seconds. The solid product was collected by filtration,and dried in a vacuum oven (50° C.), to afford the compound EO (2.7 g,65%). ¹H NMR (D₂O, 500 MHz) δ ppm 7.36 (d, 1H, J=7.4 Hz), 7.24 (m, 3H),4.70 (q, 1H, J=5.5 Hz), 4.53 (d, 1H, J=5.4 Hz), 3.23 (t, 2H, J=7.8 Hz),3.10 (m, 1H), 2.89 (m, 3H), (m, 1H), 2.08 (m, 2H). ¹³C NMR (D₂O, 125MHz) δ ppm 141.60, 134.30, 130.53, 127.61, 126.10, 125.69, 70.42, 64.08,48.25, 44.73, 38.33, 21.50. [α]_(D)=+3.0° (c=0.0018, water) ES-MS 272(M+1).

Preparation of3-[(1S)-1-(hydroxymethyl)-2-methylpropyl]amino-1-propanesulfonic acid(Compound EP)

To a solution of (S)-(−)-2-amino-3-methyl-1-butanol (2.50 g, 24.2 mmol)in tetrahydrofuran (35 mL) was slowly added 1,3-propane sultone (2.89 g,23.0 mmol). The mixture was stirred at reflux for 3 h. The reactionmixture was cooled to room temperature. The solid material was collectedby filtration, washed with acetone (2×25 mL). The crude product wassuspended in 80% acetone/ethanol (75 mL). The suspension was stirred atreflux for 30 seconds. The solid product was collected by filtration,and dried in a vacuum oven (50° C.), to afford compound EP (2.9 g, 56%).¹H NMR (D₂O, 500 MHz) δ ppm 3.78 (dd, 1H), 3.62 (dd, 1H), 3.13 (m, 2H),2.90 (m, 1H), 2.88 (t, 3H), 1.90 (m, 3H), 0.90 (d, 3H), 0.84 (d, 3H).¹³C NMR (D₂O, 125 MHz) δ ppm 64.74, 57.24, 48.20, 44.44, 26.98, 21.49,18.53, 17.00. [α]_(D)=+4.1° (c=0.0017 in water), ES-MS 224 (M−1).

Preparation of3-[(1S)-1-carbamoyl-2-methylpropyl]amino-1-propanesulfonic acid(Compound EQ)

L-valinamide hydrochloride (2.50 g, 16.4 mmol) was treated with asaturated solution of K₂CO₃ (75 mL). The mixture was extracted withEtOAc (3×75 mL). The organic extracts were combined, dried over Na₂SO₄.The solid material was removed by filtration, and the filtrate wasconcentrated to dryness under reduced pressure. The residual materialwas dried in vacuo.

To a solution of L-valinamide (1.57 g, 13.5 mmol) in tetrahydrofuran (20mL) was slowly added 1,3-propane sultone (1.61 g, 12.9 mmol). Themixture was stirred at reflux for 2 hours. The reaction mixture wascooled to room temperature. The solid material was collected byfiltration, washed with acetone (2×25 mL). The crude product wasdissolved in water (60 mL) and treated with ion exchange resin DowexMarathon C (strongly acidic, 15 g). The mixture was stirred for 15minutes. The resin was removed by filtration. The filtrate was pouredinto EtOH (250 mL). The solid product, after the completion of theprecipitation, was collected by filtration, and dried in vacuo, toafford compound EQ (1.6 g, 51%). ¹H NMR (D₂O, 500 MHz) δ ppm 3.63 (d,1H), 3.05 (m, 2H), 2.85 (m, 2H), 2.05 (m, 4H), 0.93 (d, 3H), 0.88 (d,3H). ¹³C NMR (D₂O, 125 MHz) δ ppm 170.24, 65.92, 48.16, 46.31, 29.59,21.31, 18.02, 17.07. [α]_(D)=+10.5° (c=0.0027 in water), ES-MS 237(M−1).

Preparation of 3-isobutylamino-1-propanesulfonic acid (Compound CE)

Isobutylamine (2.4 mL, 24 mmol) was added to a solution of 1,3-propanesultone (3.04 g, 24.5 mmol) in 2-butanone (20 mL). The mixture washeated to reflux. After about 10 minutes, the mixture had turned into alump. It was cooled to room temperature. Acetone was added and the lumpwas crushed. The solid was collected by filtration and dried in-vacuo(2.2 g). The white solid was suspended in ethanol (10 mL) and themixture brought to reflux. A significant amount of the solid dissolved.Water was added slowly until a clear pink solution was obtained. Thesolution was left at room temperature overnight. The flask was placed ina fridge for 2 hours. The solid product was collected by filtration,rinsed with ethanol (5 mL), washed with ether (10 mL), and driedin-vacuo. Compound CE was obtained as long, fine white needles (1.87 g,40% yield). m.p. 255-57° C. ¹H NMR (500 MHz, D₂O) δ 0.88 (d, J=6.8 Hz,6H), 1.85-1.93 (m, J=6.8 Hz, 1H), 2.03 (qt, J=7.6 Hz, 2H), 2.80 (d,J=7.3 Hz, 2H), 2.90 (t, J=7.3 Hz, 2H), 3.08 (t, J=8.1 Hz, 2H). ¹³C NMR(125 MHz, D₂O) δ 19.2, 21.2, 25.8, 46.9, 48.1, 54.9 ES-MS 196 (M+1).FT-IR (KBr) ν_(max) 3566, 2973, 2021, 1719.

Preparation of 3-isoamylamino-1-propanesulfonic acid (Compound CH)

Isoamylamine (4 mL, 34.5 mmol) was added to a solution of 1,3-propanesultone (4.7 g, 38 mmol) in 2-butanone (70 mL). The mixture was warmedto reflux. After 30 minutes, the mixture was too thick to stir. Acetone(15 mL) was added. The reflux was maintained for a total of 4 hours. Thesuspension was cooled at room temperature. The white solid was collectedby filtration, rinsed with acetone (10 mL), then with ether (10 mL).Compound CH was obtained as a very light, fluffy white solid (4.48 g,62% yield). m.p. 220° C.: decomposed. ¹H NMR (500 MHz, D₂O) δ 0.65 (d,J=6.3 Hz, 6H), 1.29 (q, J=7.7 Hz, 2H), 1.35-1.42 (m, J=6.6 Hz, 1H), 1.86(qt, J=7.6 Hz, 2H), 2.74 (t, J=7.3 Hz, 2H), 2.81 (t, J=8.1 Hz, 2H), 2.93(t, J=7.8 Hz, 2H). ¹³C NMR (125 MHz, D₂O) δ 21.3, 21.4, 25.2, 34.2,46.1, 46.2, 47.9

Preparation of 2-(tert-butyl)amino-1-ethanesulfonic Acid (Compound DU)

A solution of 2-bromoethanesulfonic acid, sodium salt (4.2 g, 20 mmol)in water (total 12 mL) was added over 6 hours to a 42° C. solution oft-butylamine (10 mL, 94 mmol) in a mixture of water (10 mL) and1,4-dioxane (10 mL). The mixture was stirred at 42° for 18 hours. Themixture was then heated to 60° C. for 24 h. By proton NMR, 30% ofelimination product (vinylsulfonic acid) was observed. The mixture wasconcentrated to dryness and treated with ethanol at refluxingtemperature. The solid material was collected (crop 1). The motherliquor was concentrated to dryness and the solid was again treated withethanol at refluxing temperature, and the solid material was collected(crop 2). Both crops of the solid material were dissolved in water, andthe resultant aqueous solutions passed in sequence through a Dowex 50 WX 8 ion-exchange column (100 g resin). The fractions containing thetitle compound were collected and concentrated to dryness. The solidmaterial obtained was recrystallized from a mixture of ethanol (20 mL)and water (2 mL). The crystals were collected by filtration, dried in avacuum oven at 60° C. for 18 hours. Compound DU was obtained as finewhite needles (860 mg, 24% yield). ¹H NMR (500 MHz, D₂O) δ 1.16 (s, 9H),3.02 (t, J=6.8 Hz, 2H), 3.19 (t, J=6.8 Hz, 2H). ¹³C NMR (125 MHz, D₂O) δ24.8, 37.3, 47.0, 57.8. ES-MS 182 (M+1)

Preparation of 3-(cyclohexanemethyl)amino-1-propanesulfonic acid(Compound DV)

A mixture of cyclohexanemethylamine (11.12 mL, 0.085 mol) and1,3-propane sultone (11.00 g, 0.090 mol) in acetonitrile (120 mL) washeated at reflux for 2 hours. The mixture was cooled to roomtemperature. The solid was collected by filtration, air-dried for 20minutes (19 g). The solid was suspended in methanol (100 mL) and thesuspension was heat to reflux. Water (4 mL) was added dropwise until aclear solution was obtained at refluxing temperature. The mixture wasthen cooled to 5° C. with stirring. The solid was collected by suctionfiltration, air-dried for 45 minutes, and further dried in a vacuum ovenat 60° C. for over 3 days. Compound DV was obtained as white flakes,(16.23 g, 81% yield.). ¹H NMR (500 MHz, D₂O) δ 0.82 (br q, J=11 Hz, 2H),0.91-1.09 (m, 3H), 1.43-1.53 (m, 6H), 1.93 (qt, J=7.3 Hz, 2H), 2.71 (d,J=6.3 Hz, 2H), 2.80 (t, J=7.3 Hz, 2H), 2.71 (t, J=7.8 Hz, 2H). ¹³C NMR(125 MHz, D₂O) δ 21.2, 25.0, 25.5, 29.9, 34.7, 46.8, 48.1, 53.7. ES-MS236 (M+1)

Preparation of 3-(1,1-diethylpropargyl)amino-1-propanesulfonic acid(Compound DW)

A mixture of 1,1-diethylpropargylamine (5 g, 45 mmol) and 1,3-propanesultone (6.05 g, 49.5 mmol) in THF (25 mL) was heated at reflux for 5hours. The mixture was cooled to room temperature. The solid wascollected by filtration, rinsed with diisopropylether (2×10 mL) thendried overnight in the vacuum oven (7.16 g). The solid was suspended inethanol (30 mL) and the suspension was heated at reflux for 1 hour. Themixture was then cooled to room temperature and the solid was collectedby suction filtration, air-dried for 5 m, and further dried in a vacuumoven at 60° C. overnight (5.86 g). There was still a significant amountof ethanol present. The solid was further dried in the vacuum oven for40 hours. Compound DW was obtained as a fine white solid, (5.66 g, 81%yield). ¹H NMR (500 MHz, D₂O) δ 0.92 (t, J=7.6 Hz, 2H), 1.71 (q, J=7.3Hz, 3H), 1.93 (qt, J=7.3 Hz, 2H), 2.81 (t, J=7.3 Hz, 2H), 2.94 (s, 1H),3.13 (t, J=7.6 Hz, 2H). ¹³C NMR (125 MHz, D₂O) δ 7.2, 21.6, 27.8, 41.4,48.1, 62.2, 78.6, 78.9. ES-MS 234 (M+1)

Preparation of 3-(1-ethynylcyclohexyl)amino-1-propanesulfonic acid(Compound DX)

A mixture of 1-ethynylcyclohexylamine (6 g, 48.7 mmol) and 1,3-propanesultone (6.55 g, 53.6 mmol) in THF (35 mL) was heated at reflux for 2hours (thick paste). The mixture was cooled to room temperature. Thesolid was collected by filtration, rinsed with THF (3×5 mL), air-dried15 minutes (7.3 g). The solid was suspended in ethanol (30 mL) and thesuspension was heated at reflux for 1 hour. The mixture was then cooledto room temperature and the solid was collected by suction filtration,rinsed with ethanol (2×5 mL), air-dried for 10 min, and further dried ina vacuum oven at 60° C. overnight (crop1, 7.10 g). The combined motherliquors were stirred overnight at room temperature. There was a lot ofsolid. The solid was collected by filtration, rinsed with acetone (3×5mL), air-dried for 30 minutes, and then suspended in ethanol (12 mL).The suspension was heated at reflux for 1 hour. The mixture was thencooled to room temperature and the solid was collected by suctionfiltration, rinsed with ethanol (2×5 mL), air-dried for 2 minutes, andfurther dried in a vacuum oven at 60° C. overnight (crop 2: 1.85 g).Compound DX was obtained as a fine white solid (two crops in total 8.95g, 75% yield). ¹H NMR (500 MHz, D₂O) δ 0.95-1.05 (m, 1H), 1.38-1.54 (m,5H), 1.60-1.64 (m, 2H), 1.94 (qt, J=7.8 Hz, 2H), 2.85 (t, J=7.3 Hz, 2H),3.01 (s, 1H), 3.22 (t, J=7.8 Hz, 2H). ¹³C NMR (125 MHz, D₂O) δ 21.8,22.3, 24.2, 34.4, 40.9, 48.0, 59.2, 78.6, 79.3. ES-MS 243.0 (M−1).

Preparation of (3-(2-hydroxy-2-phenyl)amino-1-propanesulfonic acid(Compound DY)

A mixture of (±)-2-Amino-1-phenylethanol (9.9 g, 72 mmol) and1,3-propane sultone (9.3 g, 76 mmol) in acetonitrile (70 mL) and ethanol(2 mL) was heated at reflux for 1.5 hours. The mixture was cooled toroom temperature. The solid was collected by filtration, rinsed withacetonitrile (2×25 mL) air-dried for 20 minutes (21.3 g). The solid wassuspended in methanol (110 mL) and the suspension was heated to reflux.Water (4 mL) was added dropwise until a clear solution was obtained. Themixture was then cooled to room temperature. The solid was collected bysuction filtration, air-dried for 30 minutes, and further dried in avacuum oven at 60° C. for 40 hours (crop 1, 4.47 g). The combined motherliquor was stored at −20° C. for 40 hours. A second crop of the solidwas collected by filtration, rinsed with acetone (2×15 mL), air-dried (1hour), and further dried in a vacuum oven at 60° C. for 24 hours.Compound DY was obtained in two crops (total 7.82 g, 42% yield). ¹H NMR(500 MHz, D₂O) 8, 2.03-2.06 (m, 2H), 2.90 (t, J=7.3 Hz, 2H), 3.16 (t,J=7.3 Hz, 2H), 3.20-3.24 (m, 2H), 4.92-4.95 (m, 1H), 7.30-7.37 (m, 5H).¹³C NMR (125 MHz, D₂O) δ 21.3, 46.6, 78.1, 53.2, 69.0, 126.1, 129.0,129.2, 139.6. ES-MS 260 (M+1).

Preparation of 3-[(S)-1-(4-methoxyphenyl)ethyl]amino-1-propanesulfonicacid (Compound DZ)

A mixture of (S)-(−)-(4-methoxyphenyl)ethylamine (1.83 g, 12.1 mmol) and1,3-propane sultone (1.6 g, 13 mmol) in acetonitrile (25 mL) was heatedat reflux for 2.5 hours. The mixture was cooled to room temperature. Thesolid was collected by filtration, rinsed with acetonitrile (2×5 mL)air-dried for 15 minutes (3.07 g). The solid was suspended in ethanol(15 mL) and the suspension was heated at reflux for 1 hour. The mixturewas then cooled to room temperature. The solid was collected by suctionfiltration, rinsed with ethanol (2×10 mL), air-dried for 15 minutes, andfurther dried in a vacuum oven at 60° C. for 18 hours. Compound DZ wasobtained as a white solid (2.95 g, 10.8 mmol, 89% yield). ¹H NMR (500MHz, D₂O) δ 1.52 (d, J=6.8 Hz, 3H), 1.88-1.98 (m, 2H), 2.76-2.79 (m,3H), 2.80-2.98 (m, 1H), 3.71 (s, 3H), 4.25 (qt, J=6.7 Hz, 2H), 6.93 (d,J=8.3 Hz, 2H), 7.29 (d, J=8.3 Hz, 2H). ¹³C NMR (125 MHz, D₂O) δ 18.3,21.5, 44.2, 48.1, 55.6, 58.0, 114.9, 128.2, 129.4, 159.9. ES-MS 274.(M+1). [α]_(D)=−28.8° (c=0.0038 in water)

Preparation of 3-(4-bromophenethyl)amino-1-propanesulfonic acid(Compound EA)

A mixture of 4-Bromophenethylamine (4 g, 20 mmol) and 1,3-propanesultone (2.56 g, 21 mmol) in acetonitrile (30 mL) was heated at refluxfor 2.5 hours. The mixture was cooled to room temperature. The solid wascollected by filtration, rinsed with acetonitrile (2×5 mL) air-dried for15 minutes (9.57 g), and further dried for 15 minutes in vacuo (8.02 g).The solid was suspended in ethanol (40 mL) and the suspension was heatedat reflux for 1 hour. The mixture was then cooled to room temperature.The solid was collected by suction filtration, rinsed with ethanol (2×5mL), air-dried for 15 minutes, and further dried in a vacuum oven at 60°C. for 18 hours. Compound EA was obtained as a white solid (6.04 g, 18.8mmol, 94% yield). ¹H NMR (500 MHz, DMSO) δ 1.95 (t, J=6.3 Hz, 3H), 2.63(t, J=6.1 Hz, 2H), 2.88 (t, J=7.6 Hz, 2H), 3.09 (t, J=6.3 Hz, 2H), 3.15(t, J=7.8 Hz, 2H), 7.25 (2, J=7.8 Hz, 2H), 7.53 (2, J=7.8 Hz, 2H), 8.63(br s, 2H). ¹³C NMR (125 MHz, DMSO) δ 21.8, 31.0, 46.7, 47.2, 48.8,119.9, 131.0, 131.4, 136.5. ES-MS 324 (M+1).

Preparation of 3-[(S)-1-indanamino]-1-propanesulfonic acid (Compound EB)

A mixture of (S)-(−)-1-aminoindan (0.92 g, 6.9 mmol) and 1,3-propanesultone (0.93 g, 7.6 mmol) in acetonitrile (15 mL) was heated at refluxfor 2.5 hours. The mixture was cooled to room temperature. The solid wascollected by filtration, rinsed with acetonitrile (2×4 mL), air-driedfor 15 minutes. The solid was suspended in ethanol (12 mL) and thesuspension was heated at reflux for 1 hour. The mixture was then cooledto room temperature. The solid was collected by suction filtration,rinsed with ethanol (2×4 mL), air-dried for 15 minutes, and furtherdried in a vacuum oven at 60° C. over the weekend. Compound EB wasobtained as a light pink solid (1.54 g, 87% yield). ¹H NMR (500 MHz,D₂O) δ 2.00 (qt, J=7.3 Hz, 2H), 2.09-2.13 (m, 1H), 2.40-2.45 (m, 1H),2.84-2.87 (m, 3H), 2.98-3.04 (m, 1H), 3.12 (t, J=7.8 Hz, 2H), 4.67-4.70(m, 1H), 7.22 (m, 2H), 7.29-7.32 (m, 2H), 7.40 (d, J=7.8 Hz, 1H). ¹³CNMR (125 MHz, D₂O) δ 21.6, 28.6, 29.8, 43.9, 48.1, 63.0, 125.5, 125.7,127.2, 130.3, 136.3, 145.4. ES-MS 256 (M+1). [α]_(D)=−1.0° (c=0.003095in water).

Preparation of 3-cyclobutylamino-1-propanesulfonic acid (Compound EC)

A mixture of cyclobutylamine (1.11 g, 15.6 mmol) and 1,3-propane sultone(2 g, 17 mmol) in acetonitrile (18 mL) was heated at reflux. The mixtureturned to a lump within 15 minutes. THF (10 mL) was added. The refluxwas maintained for 1 hour. The mixture was cooled to room temperature.The solid was collected by filtration, rinsed with acetonitrile (2×4mL), air-dried for 60 minutes (2.41 g). The solid was suspended inmethanol (20 mL) and the suspension was heated at reflux until all thesolid material was dissolved. The mixture was then cooled to roomtemperature. The solid thus formed was collected by suction filtration,rinsed with methanol (2×4 mL), air-dried for 20 minutes, and furtherdried in a vacuum oven at 40° C. for 18 hours. Compound EC was obtainedas a white solid (1.81 g, 60% yield). ¹H NMR (500 MHz, D₂O) δ 1.70-1.77(m, 2H), 1.94-2.03 (m, 4H), 2.18 (br s, 2H), 2.85 (t, J=6.8 Hz, 1H),2.95 (t, J=7.3 Hz, 1H), 3.63-3.66 (m, 1H). ¹³C NMR (125 MHz, D₂O) δ14.5, 21.5, 26.1, 43.6, 48.0, 51.8. ES-MS 194 (M+1).

Preparation of 3-(4-mexiletino)-1-propanesulfonic acid (Compound EV)

Mexiletine hydrochloride (2.45 g, 11.3 mmol) was freed with 1N NaOH (50mL), extracted with ethyl acetate (2×50 mL). The combined extract wasdried over sodium sulfate. The solvent was evaporated. A solution of1,3-propane sultone (1.46 g, 11.9 mmol) in THF (35 mL) was added to thefree amine. The mixture was heated at reflux for 4 hours. The mixturewas cooled to room temperature; and the resultant solid material wascollected by filtration, rinsed with THF (5 mL). The solid was driedovernight at 40° C. The filtrated dried in air overnight to afford abrownish solid (1.45 g) it was less pure than the first crop thusdiscarded. Compound EV was obtained as a white solid (1.19 g, 56% (99%crude) yield). ¹H NMR (500 MHz, DMSO) δ 1.39 (d, J=6.3 Hz, 3H), 2.02 (t,J=5.9 Hz, 2H), 2.26 (s, 6H), 2.67 (t, J=5.9 Hz, 2H), 3.21 (br d, J=19.5Hz, 2H), 3.61 (br s, 1H), 3.83-3.91 (m, 2H), 6.95 (t, J=7.3 Hz, 1H),7.04 (m, 2H), 8.91 (br s, 2H). ¹³C NMR (125 MHz, DMSO) δ 13.3, 16.0,21.8, 44.6, 49.2, 52.9, 70.8, 124.3, 128.9, 130.4, 154.2. ES-MS 302(M+1).

Preparation of 3-(1-benzyl-2-methoxyethyl))amino-1-propanesulfonic acid(Compound EW)

S-(+)-2-Amino-1-methoxy-3-phenylpropane hydrochloride (2.06 g, 10.0mmol) was freed with saturated potassium carbonate (20 mL). The aqueousmixture was extracted with ethyl acetate (3×15 mL); and the combinedextract was dried over sodium sulfate. The solvent was evaporated. Asolution of 1,3-propane sultone (1.29 g, 10.5 mmol) in THF (15 mL) wasadded to the free amine. The mixture was heated at reflux for 4 hours.The mixture was cooled to room temperature and stirred for 1 hour. Thesolid was collected by filtration, rinsed with acetone (5 mL). The solidwas dried overnight at 40° C. Compound EW was obtained as a white solid(2.48 g, 83% yield). ¹H NMR (500 MHz, DMSO) δ 1.99 (m, 2H), 2.65 (t,J=6.1 Hz, 2H), 2.80 (t, J=12.0 Hz, 1H), 3.05 (m, 2H), 3.17 (m, 3H), 3.22(dd, J=3.4 Hz, 10.7 Hz, 2H), 3.33 (m, 1H), 3.43 (d, J=10.7 Hz, 2H), 3.50(m, 1H), 7.27 (m, 3H), 7.35 (t, J=7.1 Hz, 2H), 8.86 (br d, 1H). ¹³C NMR(125 MHz, DMSO) δ 21.8, 33.4, 45.0, 49.2, 57.8, 58.5, 68.1, 126.9,128.7, 129.2, 136.3. ES-MS 310 (M+1). [α]=−0.8° (c=0.0025 in water).

Preparation of3-1-(N-hydroxycarbamoyl)-2-phenylethyl)amino-1-propanesulfonic acid(Compound FO)

A solution of hydroxylamine 50% in water (wt./wt, 7 mL) was added to asolution of L-N-(3-sulfopropyl)phenylalanine ethyl ester (1.00 g, 3.17mmol in water (5 mL). The mixture was stirred at room temperature for 24hours. The mixture was concentrated to dryness. The resulting solid wasdissolved in a mixture of hot methanol (10 mL) and water; and themixture was stored at 5° C. for 3 days. Only a very small amount ofsolid had formed. Upon addition of acetone (3 mL), a large amount ofsolid formed. The solid was collected by suction-filtration, rinsed withacetone (2×5 mL) then dried overnight at 40° C. in a vacuum oven.Compound FO was obtained as a white solid (700 mg, 73%). ¹H NMR (500MHz, D₂O) δ 2.03-2.07 (m, 2H), 2.88-2.90 (M, 2H), 2.99-3.03 (M, 1H),3.07-3.12 (M, 1H), 3.18-3.23 (m, 1H), 3.82-3.85 (m, 1H), 7.15 (d, J=6.8Hz, 2H), 7.26-7.31 (m, 3H). ¹³C (125 MHz, D₂O) δ 19.3, 33.8, 43.2, 45.8,57.9, 126.0, 127.1, 127.3, 131.4, 162.2. ES-MS 303 (M+Na). [α]_(D)=40°(c=0.001983 in water).

Preparation of3-{[(1S)-1-benzyl-2-(benzyloxy)-2-oxoethyl]amino}propane-1-sulfonic acid(Compound EL)

L-Phenylalanine benzylester hydrochloride (2.0 g, 6.9 mmol) was treatedwith a saturated aqueous solution of K₂CO₃ (50 mL) and EtOAc (3×50 mL)was added. The organic extracts were separated, combined, dried withNa₂SO₄, filtered, evaporated under reduced pressure and dried in vacuo.

To a solution of L-Phenylalanine benzylester (1.8 g, 6.8 mmol) in1,4-dioxane (10 mL) was added 1,3-propanesultone (708 mg, 6.5 mmol). Thesolution was stirred at reflux. After 1 hour, 20 mL of 1,4-dioxane wasadded to allow for good stiffing. The reaction was stirred at reflux foran additional 1 hour. It was cooled to room temperature. The solid wascollected by filtration, washed with acetone (2×25 mL). The product wassuspended 80% Acetone/EtOH. The suspension was stirred at reflux for 30seconds. The solid was filtered and dried in the vacuum oven (50° C.)affording the title compound (1.14 g, 46%). ¹H NMR (DMSO, 500 MHz) δ ppm7.27 (m, 6H), 7.12 (m, 4H), 5.05 (dd, 2H, J=12.3 Hz), 4.49 (m, 1H). 3.29(m, 1H), 3.00 (m, 1H), 2.98 (m, 1H), 2.61 (t, 2H, J=6.5 Hz), 1.97 (m,2H). ¹³C (DMSO, 125 MHz) δ ppm 168.88, 135.21, 134.90, 130.00, 129.31,129.07, 128.01, 67.98, 60.50, 49.77, 46.72, 35.87, 22.43. [α]_(D)=+4.8°(c=0.00073 in water), ES-MS 378 (M+1).

Preparation of3-{[(1S)-1-(methoxycarbonyl)-2-methylpropyl]amino}-1-propanesulfonicacid (Compound FT)

L-valine methylester hydrochloride (5.0 g, 29.8 mmol) was treated with asaturated solution of K₂CO₃ (75 mL) and EtOAc (3×75 mL) was added. Theorganic extracts were separated, combined, dried with Na₂SO₄, filtered,evaporated under reduced pressure and dried in vacuo.

To a solution of L-valine methylester in THF (25 mL) was slowly added1,3-propanesultone (2.49 g, 19.9 mmol). The solution was stirred atreflux for 2 hours. The reaction was cooled to room temperature. Thesolid was collected by filtration, washed with acetone (2×30 mL). It wasdried in the vacuum oven (50° C.) affording the title compound (2.52 g,50%). ¹H NMR (D₂O, 500 MHz) δ ppm 3.92 (m, 1H), 3.75 (s, 3H), 3.13 (t,2H, J=6.8 Hz), 2.88 (t, 2H, J=6.8 Hz), 2.24 (m, 1H), 2.06 (m, 2H)), 0.96(d, 3H, J=6.8 Hz), 0.88 (d, 3H, J=6.8 Hz). ¹³C (D₂O, 125 MHz) δ ppm169.35, 65.85, 55.61, 48.14, 45.59, 29.48, 21.32, 18.25, 16.57.[α]_(D)=+9.6° (c=0.0014 in water), ES-MS 254 (M+1).

Preparation of 3-{[(1R)-1-cyclohexylethyl]amino}-1-propanesulfonic acid(Compound FU)

To a solution of (R)-(−)-cyclohexylethylamine (2.5 g, 19.7 mmol) intetrahydrofuran (25 mL) was slowly added 1,3-propanesultone (2.33 g,18.7 mmol). The solution was stirred at reflux for 2 hours. The reactionwas cooled to room temperature. The solid was collected by filtration,washed with acetone (2×25 mL) and dried in vacuo, affording the titlecompound (3.47 g, 74%). ¹H NMR (D₂O, 500 MHz) δ ppm 3.09 (m, 3H), 2.88(t, 2H, J=7.3 Hz), 2.00 (m, 2H), 1.58 (m, 6H), 1.13 (m, 5H), 1.03 (m,3H). ¹³C (D₂O, 125 MHz) δ ppm 58.37, 48.17, 44.00, 39.84, 29.00, 26.01,25.82, 25.73, 25.47, 21.51, 11.79. [α]_(D)=+4.5° (c=0.0022 in water),ES-MS 250 (M−1).

Preparation of 3-{[(1S)-1-cyclohexylethyl]amino}-1-propanesulfonic acid(Compound FW)

To a solution of (S)-(+)-cyclohexylethylamine (5.0 g, 39.3 mmol) intetrahydrofuran (50 mL) was slowly added 1,3-propanesultone (4.66 g,37.4 mmol). The solution was stirred at reflux for 2 hours. The reactionwas cooled to room temperature. The solid was collected by filtrationand washed with acetone (2×25 mL). The solid was suspended in 80%acetone/EtOH (200 mL). The suspension was stirred at reflux for 30seconds before the solid was filtered and dried in vacuo, affording thetitle compound (6.13 g, 66%). ¹H NMR (D₂O, 500 MHz) δ ppm 3.09 (m, 3H),2.88 (t, 2H, J=7.3 Hz), 2.00 (m, 2H), 1.55 (m, 6H), 1.13 (m, 5H), 1.03(m, 3H). ¹³C (D₂O, 125 MHz) δ ppm 59.37, 48.17, 44.00, 39.84, 29.00,26.01, 25.82, 25.73, 25.47, 21.51, 11.78. [α]_(D)=−2.8° (c=0.0014 inwater), ES-MS 250 (M−1).

Preparation of 3-[(4-tert-butylcyclohexyl)amino]-1-propanesulfonic acid(Compound FX)

To a solution of 4-tert-butylcyclohexylethylamine (mixture of cis andtrans isomers, 2.5 g, 16.1 mmol) in tetrahydrofuran (30 mL) was slowlyadded 1,3-propanesultone (1.84 g, 15.3 mmol). The solution was stirredat reflux for 2 hours. The reaction was cooled to room temperature. Thesolid was collected by filtration and washed with acetone (2×35 mL). Thesolid was suspended in 80% acetone/EtOH (200 mL). The suspension wasstirred at reflux for 30 seconds before the solid was filtered and driedin vacuo, affording the title compound (3.07 g, 72%). ¹H NMR (DMSO, 500MHz) δ ppm 3.21 (m, 0.5H), 3.04 (m, 2H), 2.89 (m, 1H), 2.67 (m, 0.5H),1.97 (m, 4H), 1.77 (m, 1H), 1.52 (m, 1H), 1.19 (m, 2H), 0.96 (m, 2H),0.81 (s, 9H). ¹³C (DMSO, 125 MHz) δ ppm 56.47, 53.62, 50.44, 49.77,47.56, 47.04, 46.28, 44.49, 32.98, 32.74, 29.61, 28.10, 28.03, 25.57,22.68, 22.48, 21.01. ES-MS 276 (M−1).

Preparation of3-{[(1S,2S)-2-(benzyloxy)cyclopentyl]amino}-1-propanesulfonic acid(Compound FY)

To a solution of (1S,2S)-2-benzyloxycyclopentylamine (1.0 g, 5.2 mmol)in tetrahydrofuran (12 mL) was slowly added 1,3-propanesultone (601 mg,5.0 mmol). The solution was stirred at reflux for 2 hours. The reactionwas cooled to room temperature. The solid was collected by filtration,washed with acetone (2×25 mL) and dried in vacuo, affording the titlecompound (1.36 g, 87%). ¹H NMR (D₂O, 500 MHz) δ ppm 7.32 (m, 5H), 4.53(d, 1H, J=11.2 Hz), 4.41 (d, 1H, J=11.2 Hz), 4.01 (m, 1H), 3.36 (m, 1H),3.00 (t, 2H, J=7.8 Hz), 2.80 (t, 2H, J=7.8 Hz), 2.00 (m, 4H), 1.64 (m,3H), 1.49 (m, 1H). ¹³C NMR (D₂O, 125 MHz) δ ppm 136.99, 129.06, 129.01,128.77, 81.78, 71.81, 63.88, 48.01, 45.33, 29.91, 27.43, 21.60, 20.93.[α]_(D)=+31.1° (c=0.0064 in water). ES-MS 314 (M+1).

Preparation of3-{[(1R,2R)-2-(benzyloxy)cyclopentyl]amino}-1-propanesulfonic acid(Compound FZ)

To a solution of (1R,2R)-2-benzyloxycyclopentylamine (1.0 g, 5.2 mmol)in tetrahydrofuran (12 mL) was slowly added 1,3-propanesultone (601 mg,5.0 mmol). The solution was stirred at reflux for 2 hours. The reactionwas cooled to room temperature. The solid was collected by filtrationand washed with acetone (2×15 mL). The product was suspended in EtOH andthe solvent was evaporated (to remove THF residue). The solid was driedin vacuo, affording the title compound (717 mg, 46%). ¹H NMR (D₂O, 500MHz) δ ppm 7.32 (m, 5H), 4.53 (d, 1H, J=11.2 Hz), 4.42 (d, 1H, J=11.2Hz), 4.02 (m, 1H), 3.36 (m, 1H), 3.01 (t, 2H, J=7.8 Hz), 2.81 (t, 2H,J=7.8 Hz), 2.01 (m, 4H), 1.65 (m, 3H), 1.49 (m, 1H). ¹³C NMR (D₂O, 125MHz) δ ppm 137.00, 129.07, 129.01, 128.77, 81.78, 71.81, 63.89, 48.02,45.34, 29.93, 27.43, 21.61, 20.94. [α]_(D)=−38.8° (c=0.00122 in water).ES-MS 314 (M+1).

Preparation of3-{[(1S)-1-benzyl-2-(cyclohexylamino)-2-oxoethyl]amino}-1-propanesulfonicacid (Compound GA)

To a solution of L-Phenylalanine cyclohexylamide (2.5 g, 10.1 mmol) intetrahydrofuran (25 mL) was added 1,3-propanesultone (1.17 g, 9.7 mmol).The solution was stirred at reflux for 2 hours. It was cooled to roomtemperature. The solid was collected by filtration, washed with acetone(2×25 mL) and dried in the vacuum oven (50° C.), affording the titlecompound (1.39 g, 39%). ¹H NMR (D₂O, 500 MHz) δ ppm 7.21 (m, 3H), 7.08(m, 2H), 4.42 (m, 0.5H), 3.83 (m, 1H). 3.29 (m, 1H), 3.15 (m, 1H), 3.02(m, 2H), 2.86 (m, 3H), 2.49 (m, 0.5H), 2.01 (m, 2H), 1.54 (m, 1H), 1.45(m, 1H), 1.33 (m, 2H), 1.02 (m, 4H), 0.55 (m, 1H). ¹³C (D₂O, 125 MHz) δppm 133.71, 129.54, 129.18, 128.03, 62.08, 49.25, 47.97, 45.41, 36.29,31.73, 31.46, 24.86, 24.28, 24.20, 21.39. [α]_(D)=+36.4° (c=0.0019 inwater), ES-MS 369 (M+1).

Preparation of3-{[(1S,2S)-2-(benzyloxy)cyclopentyl]amino}-1-propanesulfonic acid(Compound GB)

To a solution of (1S,2S)-2-benzyloxycyclohexylamine (1.0 g, 5.2 mmol) intetrahydrofuran (12 mL) was slowly added 1,3-propanesultone (601 mg, 5.0mmol). The solution was stirred at reflux for 2 hours. The reaction wascooled to room temperature. The solid was collected by filtration,washed with acetone (2×20 mL) and dried in the vacuum oven (50° C.),affording the title compound (1.15 g, 75%). ¹H NMR (D₂O, 500 MHz) δ ppm7.34 (m, 5H), 4.62 (d, 1H, J=11.2 Hz), 4.42 (d, 1H, J=11.2 Hz), 3.40 (m,1H), 2.97 (m, 2H), 2.90 (m, 1H), 2.76 (t, 2H, J=6.5 Hz), 2.26 (m, 1H),1.92 (m, 3H), 1.66 (m, 2H), 1.18 (m, 4H). ¹³C NMR (D₂O, 125 MHz) δ ppm137.22, 129.34, 129.11, 128.84, 76.74, 70.26, 60.84, 48.02, 42.74,29.49, 26.43, 23.57, 23.02, 21.53. [α]_(D)=+74.8° (c=0.00207 in water).ES-MS 326 (M−1).

Preparation of3-{[(1R,2R)-2-(benzyloxy)cyclopentyl]amino}-1-propanesulfonic acid(Compound GD)

To a solution of (1R,2R)-2-benzyloxycyclohexylamine (1.0 g, 5.2 mmol) intetrahydrofuran (12 mL) was slowly added 1,3-propanesultone (601 mg, 5.0mmol). The solution was stirred at reflux for 2 hours. The reaction wascooled to room temperature. The solid was collected by filtration andwashed with acetone (2×35 mL). The solid was suspended in 80%acetone/EtOH (200 mL). The suspension was stirred at reflux for 30seconds before the solid was filtered and dried in the vacuum oven (50°C.), affording the title compound (844 mg. 55%). ¹H NMR (D₂O, 500 MHz) δppm 7.32 (m, 5H), 4.60 (d, 1H, J=11.2 Hz), 4.40 (d, 1H, J=11.2 Hz), 3.39(m, 1H), 2.94 (m, 2H), 2.85 (m, 1H), 2.74 (t, 2H, J=6.5 Hz), 2.24 (m,1H), 1.90 (m, 3H), 1.64 (m, 2H), 1.14 (m, 4H). ¹³C NMR (D₂O, 125 MHz) δppm 137.35, 129.28, 129.19, 128.90, 76.90, 70.35, 60.97, 48.12, 42.90,29.59, 26.53, 23.64, 23.09, 21.63. [α]_(D)=−68.9° (c=0.0026 in water).ES-MS 326 (M−1).

Preparation of3-({(1S)-1-[(benzyloxy)carbonyl]-2-methylpropyl}amino)-1-propanesulfonicacid (Compound GE)

L-valine benzylester p-tosylate (2.5 g, 6.6 mmol) was treated with asaturated solution of K₂CO₃ (50 mL) and EtOAc (3×50 mL). The organicextracts were separated, combined, dried with Na₂SO₄, filtered,evaporated under reduced pressure and dried in vacuo.

To a solution of L-valine benzylester in MeOH (12 mL) was slowly added1,3-propanesultone (604 mg, 5.0 mmol). The solution was stirred atreflux for 2 hours. The reaction was cooled to room temperature. Thesolid was collected by filtration, washed with cold MeOH and acetone(2×25 mL). It was dried in the vacuum oven (50° C.), affording the titlecompound (649 mg, 39%). ¹H NMR (D₂O, 500 MHz) δ ppm 7.47 (m, 5H), 5.41(d, 1H, J=11.7 Hz), 5.31 (d, 3H, J=11.7 Hz), 4.04 (m, 1H), 3.19 (m, 2H),2.95 (t, 2H, J=6.8 Hz), 2.35 (m, 1H), 2.14 (m, 2H), 1.04 (d, 3H, J=6.3Hz), 0.95 (d, 3H, J=6.3 Hz). ¹³C (D₂O, 125 MHz) δ ppm 168.75, 134.81,129.38, 129.31, 129.17, 69.00, 65.96, 48.24, 46.71, 29.67, 21.40, 18.39,16.65. [α]_(D)=−7.2° (c=0.0015 in water), ES-MS 330 (M+1).

Preparation of3-{[(1S)-2-ethoxy-1-methyl-2-oxoethyl]amino}-1-propanesulfonic acid(Compound GF)

L-alanine ethyl ester hydrochloride (2.5 g, 16.3 mmol) was treated witha saturated solution of K₂CO₃ (50 mL) and EtOAc (3×50 mL). The organicextracts were separated, combined, dried with Na₂SO₄, filtered,evaporated under reduced pressure and dried in vacuo.

To a solution of L-alanine ethyl ester (1.67 g, 14.3 mmol) intetrahydrofuran (25 mL) was slowly added 1,3-propanesultone (1.42 g,11.9 mmol). The solution was stirred at reflux for 2 hours. The reactionwas cooled to room temperature. The solid was collected by filtration,washed with acetone (2×25 mL) and dried in vacuo, affording the titlecompound (1.19 g, 42%). ¹H NMR (D₂O, 500 MHz) δ ppm 4.16 (m, 2H), 4.01(m, 1H), 3.12 (m, 2H), 2.87 (t, 2H, J=7.3 Hz), 2.01 (m, 2H), 1.43 (d,3H, J=7.3 Hz), 1.14 (t, 3H, J=6.8 Hz). ¹³C (D₂O, 125 MHz) δ ppm 170.22,63.84, 55.69, 47.94, 44.73, 21.51, 14.05, 13.25. [α]_(D)=−2.4° (c=0.0022in water), ES-MS 240 (M+1).

Preparation of (2S)-3-methyl-2-[(3-sulfopropyl)amino]butanoic acid(Compound GC)

L-valine methylester hydrochloride (5.0 g, 29.8 mmol) was treated with asaturated solution of K₂CO₃ (75 mL) and EtOAc (3×75 mL). The organicextracts were separated, combined, dried with Na₂SO₄, filtered,evaporated under reduced pressure and dried in vacuo.

To a solution of L-valine methylester in tetrahydrofuran (25 mL) wasslowly added 1,3-propanesultone (2.49 g, 19.9 mmol). The solution wasstirred at reflux for 2.5 hours. The reaction was cooled to roomtemperature. The solid was collected by filtration, washed with acetone(2×30 mL) and dried in vacuo affording the desired ester.

The ester (860 mg, 3.4 mmol) was dissolved in 2M NaOH (1.20 g of NaOHand 15 mL of water). The reaction was stirred at room temperatureovernight. Dowex Marathon C ion exchange resin (strongly acidic) wasadded to the solution. The suspension was stirred for 15 minutes beforethe resin was removed by filtration. The filtrate was evaporated underreduced pressure and dried in vacuo, affording the title compound (645mg, 79%). ¹H NMR (D₂O, 500 MHz) δ ppm 3.66 (m, 1H), 3.09 (t, 2H, J=6.3Hz), 2.86 (t, 2H, J=7.3 Hz), 2.17 (m, 1H), 2.07 (m, 2H)), 0.93 (d, 3H,J=6.8 Hz), 0.87 (d, 3H, J=6.8 Hz). ¹³C (D₂O, 125 MHz) δ ppm 171.24,66.83, 48.28, 46.77, 29.33, 21.44, 18.30, 16.98. [α]_(D)=−16.5°(c=0.0020 in water), ES-MS 238 (M−1).

Preparation of3-{[(1S)-1-(methoxycarbonyl)-3-methylbutyl]amino}-1-propanesulfonic acid(Compound GH)

L-Leucine methylester hydrochloride (5.0 g, 27.5 mmol) was treated witha saturated solution of K₂CO₃ (50 mL) and EtOAc (3×50 mL) was added. Theorganic extracts were separated, combined, dried with Na₂SO₄, filtered,evaporated under reduced pressure and dried in vacuo.

To a solution of L-valine methylester (3.74 g, 25.6 mmol) intetrahydrofuran (35 mL) was slowly added 1,3-propanesultone (2.04 g,17.2 mmol). The solution was stirred at reflux for 3 hours. The reactionwas cooled to room temperature. The solid was collected by filtration.Dowex Marathon C ion exchange resin (strongly acidic) was added to thesolution. The suspension was stirred for 15 minutes before the resin wasremoved by filtration. The filtrate was evaporated under reducedpressure. The solid was suspended in acetone (50 mL), filtered and driedin vacuo, affording the title compound (1.80 g, 39%). ¹H NMR (D₂O, 500MHz) δ ppm 3.99 (m, 1H), 3.72 (s, 3H), 3.12 (m, 2H), 2.87 (t, 2H, J=7.3Hz), 2.02 (m, 2H), 1.74 (m, 1H), 1.60 (m, 2H), 0.81 (d, 3H, J=5.4 Hz),0.87 (d, 3H, J=6.8 Hz). ¹³C (D₂O, 125 MHz) δ ppm 170.60, 58.91, 53.81,48.08, 45.50, 38.17, 24.44, 22.15, 21.59, 20.93. [α]_(D)=+13.8°(c=0.0016 in water), ES-MS 268 (M+1).

Preparation of3-({(1S)-1-[(tert-butylamino)carbonyl]-2-methylpropyl}amino)-1-propanesulfonicacid (Compound GI)

L-valine tert-butylamide hydrochloride (2.5 g, 12.0 mmol) was treatedwith a saturated solution of K₂CO₃ (50 mL) and EtOAc (3×50 mL) wasadded. The organic extracts were separated, combined, dried with Na₂SO₄,filtered, evaporated under reduced pressure and dried in vacuo.

To a solution of L-valine tert-butylamide (1.87 g, 11.0 mmol) in1,4-dioxane (20 mL) was added 1,3-propanesultone (1.07 g, 9.0 mmol). Thesolution was stirred at reflux for 5 hours. The reaction was cooled toroom temperature. The solid was collected by filtration, washed withacetone (2×30 mL) and dried in vacuo, affording the title compound (801mg, 25%). ¹H NMR (D₂O, 500 MHz) δ ppm 3.43 (m, 1H), 3.00 (m, 2H), 2.85(m, 2H), 2.03 (m, 3H), 1.21 (m, 9H), 0.92 (d, 3H, J=6.3 Hz), 0.85 (d,3H, J=6.8 Hz). ¹³C (D₂O, 125 MHz) δ ppm 166.47, 66.55, 52.56, 48.23,46.11, 29.91, 27.81, 21.29, 18.30, 17.44. [α]_(D)=−11.6° (c=0.0023 inwater), ES-MS 293 (M−1).

Preparation of3-{[(1S)-1-(hydroxymethyl)-3-methylbutyl]amino}-1-propanesulfonic acid(Compound GJ)

To a solution of L-(+)-leucinol (5.0 g, 42.8 mmol) in THF (65 mL) wasslowly added 1,3-propanesultone (4.85 g, 40.7 mmol in THF (10 mL)). Thesolution was stirred at reflux for 2 hours. The reaction was cooled toroom temperature. The solid was collected by filtration and washed withacetone (2×50 mL). The solid was dissolved in 50% water/EtOH (400 mL).Dowex Marathon C ion exchange resin (strongly acidic) was added to thesolution. The suspension was stirred for 15 minutes before the resin wasremoved by filtration. The filtrate was evaporated under reducedpressure. The solid was suspended in acetone (150 mL), filtered anddried in the vacuum oven (50° C.), affording the title compound (6.11 g,63%). ¹H NMR (D₂O, 500 MHz) δ ppm 3.77 (m, 1H), 3.59 (m, 1H), 3.23 (m,1H), 3.13 (m, 2H), 2.90 (m, 2H), 2.02 (m, 2H), 1.53 (m, 2H), 1.35 (m,1H), 0.81 (d, 3H, J=16.1 Hz). ¹³C (D₂O, 125 MHz) δ ppm 58.72, 58.00,48.17, 43.54, 35.96, 24.34, 22.53, 21.62, 20.89. [α]_(D)=+16.6°(c=0.0022 in water), ES-MS 240 (M+1).

Preparation of3-{[(1S)-1-(hydroxymethyl)-2-methylbutyl]amino}-1-propanesulfonic acid(Compound GK)

To a solution of L-(+)-isoleucinol (2.0 g, 17.1 mmol) in THF (30 mL) wasslowly added 1,3-propanesultone (1.94 g, 16.3 mmol). The solution wasstirred at reflux for 2 hours. The reaction was cooled to roomtemperature. The solid was collected by filtration and washed withacetone (2×20 mL). The solid was dissolved in 70% water/EtOH (240 mL).Dowex Marathon C ion exchange resin (strongly acidic, 15 g) was added tothe solution. The suspension was stirred for 15 minutes before the resinwas removed by filtration. The filtrate was evaporated under reducedpressure. The solid was suspended in acetone (60 mL), filtered and driedin the vacuum oven (50° C.), affording the title compound (1.70 g, 44%).¹H NMR (D₂O, 500 MHz) δ ppm 3.78 (d, 1H, J=13.1 Hz), 3.64 (m, 1H), 3.14(m, 3H), 2.03 (m, 2H), 1.75 (m, 1H), 1.32 (m, 1H), 1.17 (m, 1H), 0.79(m, 6H). ¹³C (D₂O, 125 MHz) δ ppm 63.42, 57.38, 48.27, 44.77, 33.64,25.91, 21.52, 13.31, 10.94. [α]_(D)=+20.4° (c=0.00212 in water), ES-MS240 (M+1).

Preparation of3-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}-1-propanesulfonic acid(Compound GL)

To a solution of D-(−)-leucinol (2.0 g, 17.1 mmol) in THF (30 mL) wasslowly added 1,3-propanesultone (1.94 g, 16.3 mmol). The solution wasstirred at reflux for 2 hours. The reaction was cooled to roomtemperature. The solid was collected by filtration and washed withacetone (2×20 mL). The solid was dissolved in 50% water/EtOH (240 mL).Dowex Marathon C ion exchange resin (strongly acidic) was added to thesolution. The suspension was stirred for 15 minutes before the resin wasremoved by filtration. The filtrate was evaporated under reducedpressure. The solid was suspended in acetone (50 mL), filtered and driedin the vacuum oven (50° C.), affording the title compound (2.55 g,(65%). ¹H NMR (D₂O, 500 MHz) δ ppm 3.74 (d, 1H, J=12.7 Hz), 3.56 (d, 1H,J=12.7 Hz), 3.20 (m, 1H), 3.10 (t, 2H, J=7.3 Hz), 2.87 (t, 2H, J=7.3Hz), 2.00 (m, 2H), 1.49 (m, 2H), 1.31 (m, 1H), 0.80 (d, 3H, J=6.3 Hz),0.76 (d, 3H, J=6.3 Hz). ¹³C (D₂O, 125 MHz) δ ppm 58.74, 58.01, 48.19,43.56, 35.98, 24.34, 22.54, 21.63, 20.90. [α]_(D)=−16.3° (c=0.0019 inwater), ES-MS 238 (M−1).

Preparation of3-{[(1S)-2-amino-2-oxo-1-phenylethyl]amino}-1-propanesulfonic acid(Compound GN)

L-Phenylglycine amide hydrochloride (1.0 g, 6.7 mmol) was treated with asolution of K₂CO₃ (20 mL). The resultant mixture was extracted withEtOAc (3×20 mL). The organic extracts were separated, combined, driedover Na₂SO₄. The solid material was removed by filtration, and thefiltrate was concentrated to dryness under reduced pressure.

To a solution of L-Phenylglycine amide (670 mg, 5.9 mmol) intetrahydrofuran (10 mL) and 1,4-dioxane (4 mL) was added1,3-propanesultone (674 mg, 5.6 mmol). The solution stirred at refluxfor 2 hours. The reaction was cooled to room temperature. The solid wascollected by filtration and washed with acetone (2×20 mL). The solid wasdissolved in 50% water/EtOH mL). Dowex Marathon C ion exchange resin(strongly acidic) was added to the solution. The suspension was stirredfor 15 minutes before the resin was removed by filtration. The filtratewas evaporated under reduced pressure. The solid was suspended inacetone (50 mL), filtered and dried in the vacuum oven (50° C.),affording the title compound (743 mg, 50%). ¹H NMR (D₂O, 500 MHz) δ ppm7.38 (m, 5H), 4.92, (s, 1H), 3.01 (m, 1H), 2.91 (m, 1H), 2.78 (t, 2H,J=7.3 Hz), 2.0 (m, 2H). ¹³C (D₂O, 125 MHz) δ ppm 170.15, 130.95, 130.24,129.94, 128.74, 63.40, 47.99, 44.92, 21.27. [α]_(D)=−124° (c=0.0041 inwater), ES-MS 271 (M−1).

Preparation of3-{[(1S)-2-tert-butoxy-1-methyl-2-oxoethyl]amino}-1-propanesulfonic acid(Compound GO)

L-Alanine tert-butylester hydrochloride (2.61 g, 14.4 mmol) was treatedwith a solution of K₂CO₃ (75 mL). The resultant mixture was extractedwith EtOAc (3×75 mL). The organic extracts were separated, combined,dried over Na₂SO₄. The solid material was removed by filtration, and thefiltrate was concentrated to dryness under reduced pressure.

To a solution of L-Alanine tert-butylester (1.53 g, 10.5 mmol) intetrahydrofuran (20 mL) was added 1,3-propanesultone (1.16 g, 9.6 mmol).The solution stirred at reflux for 2 hours. The reaction was cooled toroom temperature. The solid was collected by filtration and washed withacetone (2×20 mL). The solid was dissolved in water (80 mL). DowexMarathon C ion exchange resin (strongly acidic) was added to thesolution. The suspension was stirred for 15 minutes before the resin wasremoved by filtration. The filtrate was evaporated under reducedpressure. The solid was suspended in acetone (80 mL), filtered and driedin the vacuum oven (50° C.), affording the title compound (1.37 g, 54%).¹H NMR (D₂O, 500 MHz) δ ppm 3.88 (m, 1H), 3.09 (m, 2H), 2.86 (t, 2H,J=7.3 Hz), 2.00 (m, 2H), 1.39 (d, 3H, J=7.3 Hz), 1.35 (s, 9H). ¹³C (D₂O,125 MHz) δ ppm 169.13, 86.12, 56.24, 47.94, 44.71, 27.11, 21.52, 14.17.[α]_(D)=−1.1° (c=0.0027 in water), ES-MS 266 (M−1).

Preparation of3-{[(1S)-2-amino-2-oxo-1-phenylethyl]amino}-1-propanesulfonic acid(Compound GP)

D-Phenylglycine amide hydrochloride (1.0 g, 6.7 mmol) was treated with asolution of K₂CO₃ (20 mL). The resultant mixture was extracted withEtOAc (3×20 mL). The organic extracts were separated, combined, driedover Na₂SO₄. The solid material was removed by filtration, and thefiltrate was concentrated to dryness under reduced pressure.

To a solution of D-Phenylglycine amide (850 mg, 7.5 mmol) intetrahydrofuran (10 mL) and 1,4-dioxane (4 mL) was added1,3-propanesultone (818 mg, 6.8 mmol). The solution stirred at refluxfor 2 hours. The reaction was cooled to room temperature. The solid wascollected by filtration and washed with acetone (2×20 mL). The solid wasdissolved in 50% water/EtOH mL). Dowex Marathon C ion exchange resin(strongly acidic) was added to the solution. The suspension was stirredfor 15 minutes before the resin was removed by filtration. The filtratewas evaporated under reduced pressure. The solid was suspended inacetone (50 mL), filtered and dried in the vacuum oven (50° C.),affording the title compound (720 mg, 34%). ¹H NMR (D₂O, 500 MHz) δ ppm7.38 (m, 5H), 4.92, (s, 1H), 3.00 (m, 1H), 2.90 (m, 1H), 2.78 (m, 2H),1.97 (m, 2H). ¹³C (D₂O, 125 MHz) δ ppm 170.14, 130.95, 130.24, 129.94,128.74, 63.40, 47.99, 44.92, 21.27. [α]_(D)=+106° (c=0.0016 in water),ES-MS 273 (M+1).

Preparation of (2S)-2-[(3-sulfopropyl)amino]propanoic acid (Compound GQ)

L-alanine methylester hydrochloride (5.0 g, 35.8 mmol) was treated witha saturated solution of K₂CO₃ (75 mL). The mixture was extracted withEtOAc (3×75 mL). The organic extracts were separated, combined, driedwith Na₂SO₄, filtered, evaporated under reduced pressure and dried invacuo.

To a solution of L-alanine methylester (2.37 g, 23.3 mmol) intetrahydrofuran (35 mL) was added 1,3-propanesultone (2.41 g, 20.0mmol). The solution was stirred at reflux for 2 hours. The reaction wascooled to room temperature. The solid was collected by filtration,washed with acetone (2×30 mL) and dried in vacuo.

The ester (2.21 g, 9.8 mmol) was dissolved in 2M NaOH (2.40 g of NaOHand 30 mL of water). The reaction was stirred at room temperatureovernight. Dowex Marathon C ion exchange resin (strongly acidic) wasadded to the solution. The suspension was stirred for 15 minutes beforethe resin was removed by filtration. The filtrate was evaporated underreduced pressure and lyophilized, affording the title compound (1.81 g,87%). ¹H NMR (D₂O, 500 MHz) δ ppm 3.76 (m, 1H), 3.07 (m, 2H), 2.85 (t,2H, J=7.3 Hz), 1.99 (m, 2H), 1.38 (d, 3H, J=7.3 Hz), 0.87 (d, 3H, J=6.8Hz). ¹³C (D₂O, 125 MHz) δ ppm 173.31, 56.66, 47.97, 44.76, 21.56, 14.51.[α]_(D)=+3.5° (c=0.0023 in water), ES-MS 210 (M−1).

Preparation of (2S)-3-phenyl-2-[(3-sulfopropyl)amino]propanoic acid(Compound GR)

The N-(3-sulfo-propyl)-phenylalanine ethyl ester (DM-258-069, 860 mg,2.7 mmol) was dissolved in 2N NaOH (1.2 g of NaOH and 15 mL of water).The reaction was stirred at room temperature overnight. Dowex Marathon Cion exchange resin (strongly acidic) was added to the solution. Thesuspension was stirred for 15 minutes before the resin was removed byfiltration. The filtrate was evaporated under reduced pressure andlyophilized, affording the title compound (654 mg, 84%). ¹H NMR (D₂O,500 MHz) δ ppm 7.20 (m, 5H), 3.96 (t, 1H, J=6.3 Hz), 3.11 (m, 4H), 2.80(t, 2H, J=7.3 Hz), 1.95 (m, 2H). ¹³C (D₂O, 125 MHz) δ ppm 171.46,134.03, 129.50, 129.28, 128.10, 62.02, 47.97, 45.64, 35.23, 21.39.[α]_(D)=+14.9° (c=0.0013 in water), ES-MS 286 (M−1).

Preparation of3-{[(1S)-1-isopropyl-2-oxopent-4-enyl]amino}-1-propanesulfonic acid(Compound GS)

L-Valine allylester p-tosylate (3.0 g, 9.1 mmol) was treated with asaturated solution of K₂CO₃ (30 mL). The mixture was extracted withEtOAc (3×30 mL). The organic extracts were separated, combined, driedwith Na₂SO₄, filtered and evaporated under reduced pressure.

To a solution of L-valine allylester (1.30 g, 8.3 mmol) intetrahydrofuran (6 mL), 1,4-dioxane (6 mL) and MeOH (0.5 mL) was added1,3-propanesultone (910 mg, 7.5 mmol). The solution was stirred atreflux for 2 hours. The reaction was cooled to room temperature. Thesolvent was evaporated under reduced pressure. The sticky paste wassuspended in 20% acetone/ether. The solid was filtered and dissolvedEtOH (75 mL). Dowex Marathon C ion exchange resin (strongly acidic) wasadded to the solution. The suspension was stirred for 15 minutes beforethe resin was removed by filtration. The filtrate was evaporated todryness under reduced pressure, affording the title compound (605 mg,26%). ¹H NMR (D₂O, 500 MHz) δ ppm 5.84 (m, 1H), 5.27 (d, 1H, J=17.1 Hz),5.19 (m, 1H, J=10.3 Hz), 3.91 (d, 1H, J=3.9 Hz), 3.10 (t, 2H, J=7.3 Hz),2.85 (t, 2H, J=7.3 Hz), 2.22 (m, 1H), 2.03 (m, 2H), 0.93 (d, 3H, J=6.8Hz), 0.85 (d, 3H, J=6.8 Hz). ¹³C (D₂O, 125 MHz) δ ppm 168.55, 130.90,120.31, 67.58, 65.82, 48.09, 46.57, 29.53, 21.29, 18.25, 16.57.[α]_(D)=+5.0° (c=0.0011 in water), ES-MS 278 (M−1).

Preparation of3-{[(1S)-1-(aminocarbonyl)-3-methylbutyl]amino}-1-propanesulfonic acid(Compound GT)

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L-Leucinamide hydrochloride (5.0 g, 30.0 mmol) was treated with asaturated solution of K₂CO₃ (100 mL). The mixture was extracted withEtOAc (3×100 mL). The organic extracts were separated, combined, driedwith Na₂SO₄, filtered and evaporated under reduced pressure.

To a solution of L-Leucinamide (3.20 g, 24.5 mmol) in tetrahydrofuran(35 mL) was added 1,3-propanesultone (2.82 g, 23.3 mmol). The solutionwas stirred at reflux for 2 hours. The reaction mixture was cooled toroom temperature. The solid was filtered and washed with acetone (2×25mL). The solid was dissolved in 50% EtOH/water (200 mL). Dowex MarathonC ion exchange resin (strongly acidic, 25 g) was added to the solution.The suspension was stirred for 15 minutes before the resin was removedby filtration. The filtrate was evaporated to dryness under reducedpressure. The solid was suspended in acetone (75 mL), and it was thenfiltered and dried in vacuo, affording the title compound (3.13 g, 53%).¹H NMR (D₂O, 500 MHz) δ ppm 3.79 (m, 1H), 3.04 (m, 2H), 2.85 (m, 2H),2.02 (m, 2H), 1.65 (m, 1H), 1.54 (m, 2H), 0.80 (m, 6H). ¹³C (D₂O, 125MHz) δ ppm 171.46, 59.42, 48.04, 45.46, 39.04, 24.27, 22.24, 21.49,21.17. [α]_(D)=+13.5° (c=0.0026 in water), ES-MS 251 (M−1).

Preparation of3-{[(1S)-1-(benzyloxycarbonyl)-3-methylbutyl]amino}-1-propanesulfonicacid (Compound GU)

L-Leucine benzylester p-tosylate (5.0 g, 12.7 mmol) was treated with asaturated solution of K₂CO₃ (100 mL). The mixture was extracted withEtOAc (3×100 mL). The organic extracts were separated, combined, driedwith Na₂SO₄, filtered and evaporated under reduced pressure.

To a solution of L-Leucine benzylester (2.81 g, 12.7 mmol) intetrahydrofuran (6 mL), 1,4-dioxane (6 mL) and MeOH (6 mL) was added1,3-propane sultone (1.40 g, 11.5 mmol). The solution was stirred atreflux for 2.5 hours. The reaction mixture was cooled to roomtemperature. The solid was filtered and washed with acetone (2×20 mL).The filtrate was evaporated under reduced pressure. The residue wasdissolved in acetone (20 mL). The product was precipitated with Et₂O(200 mL). The solid material was filtered. Both solids were combined anddissolved in 50% EtOH/water (200 mL). Dowex Marathon C ion exchangeresin (strongly acidic) was added to the solution. The suspension wasstirred for 15 minutes before the resin was removed by filtration. Thefiltrate was evaporated under reduced pressure and lyophilized,affording the title compound (1.87 g, 47%). ¹H NMR (DMSO, 500 MHz) δ ppm9.34 (s (broad), 1H), 7.39 (m, 5H), 5.25 (s, 2H), 4.10 (m, 1H), 3.09 (m,2H), 2.60 (m, 2H), 1.95 (m, 2H), 1.64 (m, 3H), 0.86 (m, 6H). ¹³C (DMSO,125 MHz) δ ppm 168.90, 134.91, 128.53, 128.50, 128.41, 67.38, 57.37,49.17, 45.79, 38.06, 24.07, 22.79, 21.78, 21.33. [α]_(D)=+1.8° (c=0.0017in water), ES-MS 344 (M+1).

Preparation of3-{[(1S)-1-(methyloxycarbonyl)-3-methylbutyl]amino}-1-propanesulfonicacid (Compound GZ)

L-Isoleucine methylester hydrochloride (5.0 g, 27.5 mmol) was treatedwith a saturated solution of K₂CO₃ (100 mL). The mixture was extractedwith EtOAc (3×100 mL). The organic layers were separated, combined,dried with Na₂SO₄, filtered and evaporated under reduced pressure.

To a solution of L-Isoleucine methlylester (3.43 g, 23.6 mmol) inacetone (30 mL) was added 1,3-propane sultone (2.62 g, 21.5 mmol). Thesolution was stirred at reflux for 2 h. The reaction mixture was cooledto room temperature. The solid was filtered and washed with acetone(2×20 mL). The filtrate was evaporated under reduced pressure. Theresidue was suspended in acetone (50 mL). The solid was filtered. Thesolid materials were combined and dissolved in water (100 mL). DowexMarathon C ion exchange resin (strongly acidic) was added to thesolution. The suspension was stirred for 15 minutes before the resin wasremoved by filtration. The filtrate was evaporated under reducedpressure. The solid product was suspended in acetone (100 mL), filteredand dried in vacuo, affording the title compound (3.23 g, 56%). ¹H NMR(D₂O, 500 MHz) δ ppm 4.00 (m, 1H), 3.74 (s, 3H), 3.14 (t, 2H, J=7.8 Hz),2.89 (t, 2H, J=7.3 Hz), 2.05 (m, 2H), 1.97 (m, 1H), 1.41 (m. 1H), 1.23(m, 1H), 0.83 (m, 6H). ¹³C (D₂O, 125 MHz) δ ppm 169.29, 64.51, 53.55,48.14, 46.52, 36.07, 25.92, 21.34, 13.76, 11.09. [α]_(D)=+22.6°(c=0.0023 in water), ES-MS 266 (M−1).

Preparation of3-{[(1S)-1-(oxycarbonyl)-3-methylbutyl]amino}-1-propanesulfonic acid(Compound HA)

L-Isoleucine methylester hydrochloride (5.0 g, 27.5 mmol) was treatedwith a saturated solution of K₂CO₃ (100 mL). The mixture was extractedwith EtOAc (3×100 mL). The organic layers were separated, combined,dried with Na₂SO₄, filtered and evaporated under reduced pressure.

To a solution of L-Isoleucine methlylester (3.43 g, 23.6 mmol) inacetone (30 mL) was added 1,3-propane sultone (2.62 g, 21.5 mmol). Thesolution was stirred at reflux for 2 hours. The reaction mixture wascooled to room temperature. The solid was filtered and washed withacetone (2×20 mL). The filtrate was evaporated under reduced pressure.The residue was suspended in acetone (50 mL). The solid was filtered.The solid materials were combined and dissolved in water (100 mL). DowexMarathon C ion exchange resin (strongly acidic) was added to thesolution. The suspension was stirred for 15 minutes before the resin wasremoved by filtration. The filtrate was evaporated under reducedpressure. The solid product was suspended in acetone (100 mL), filteredand dried in vacuo (3.23 g, 56%).

The solid (1.0 g, 3.7 mmol) was dissolved in 2M NaOH (30 mL). Thereaction mixture was stirred at room temperature overnight. DowexMarathon C ion exchange resin (strongly acidic, 15 g) was added to thesolution. The suspension was stirred for 15 minutes before the resin wasremoved by filtration. The filtrate was coevaporated with EtOH andlyophilized, affording the title compound (740 mg, 83%). ¹H NMR (D₂O,500 MHz) δ ppm 4.00 (m, 1H), 3.59 (m, 3H), 3.07 (t, 2H, J=7.3 Hz), 2.86(m, 2H), 2.02 (m, 2H), 1.84 (m, 1H), 1.39 (m. 1H), 1.19 (m, 1H), 0.81(m, 6H). ¹³C (D₂O, 125 MHz) δ ppm 169.29, 64.51, 53.55, 48.14, 46.52,36.07, 25.92, 21.34, 13.76, 11.09. [α]_(D)=+30.4° (c=0.0031 in water),ES-MS 252 (M−1).

Preparation of3-{[(1S)-1-carbamoyl-2-phenylethyl]amino}-1-propanesulfonic acid(Compound HB)

L-Phenylalaninamide hydrochloride (5.0 g, 24.9 mmol) was treated with asaturated solution of K₂CO₃ (75 mL). The mixture was extracted withEtOAc (3×75 mL). The organic layers were separated, combined, dried withNa₂SO₄, filtered and evaporated under reduced pressure.

To a solution of L-Phenylalaninamide (3.93 g, 23.9 mmol) in acetonitrile(25 mL) was added 1,3-propane sultone (2.70 g, 21.8 mmol). The solutionwas stirred at reflux for 2 hours. The reaction mixture was cooled toroom temperature. The solid was filtered and washed with acetonitrile(2×25 mL). The solid product was suspended in EtOH (100 mL). Thesuspension was stirred at reflux for 1 hour. The solid material wasfiltered and dried in vacuo, affording the title compound (5.18 g, 83%).¹H NMR (D₂O, 500 MHz) δ ppm 7.24 (m, 3H), 7.16 (m, 2H), 4.02 (m, 1H),3.15 (m, 1H), 3.01 (m, 3H), 2.83 (m, 2H), 2.02 (m, 2H), 1.98 (m, 2H).¹³C (D₂O, 125 MHz) δ ppm 170.48, 133.72, 129.58, 129.21, 128.11, 61.55,47.95, 45.52, 36.21, 21.44. [α]_(D)=+23.1° (c=0.0021 in water), ES-MS285 (M−1).

Preparation of3-{[(1R)-1-(methoxycarbonyl)-3-methylbutyl]amino}-1-propanesulfonic acid(Compound HC)

D-Leucine methylester hydrochloride (2.63 g, 14.5 mmol) was treated witha saturated solution of K₂CO₃ (50 mL). The aqueous mixture was extractedwith EtOAc (3×50 mL). The organic layers were separated, combined, driedwith Na₂SO₄, filtered and evaporated under reduced pressure.

To a solution of D-Leucine methylester (1.58 g, 10.9 mmol) inacetonitrile (35 mL) was added 1,3-propanesultone (1.21 g, 9.9 mmol).The solution was stirred at reflux for 2 hours. The reaction mixture wascooled to room temperature. The solid material was collected byfiltration, recrystallized from EtOH and dried in vacuo, affording thetitle compound (1.59 g, 60%). ¹H NMR (D₂O, 500 MHz) δ ppm 3.98 (m, 1H),3.70 (s, 3H), 3.11 (m, 2H), 2.85 (m, 2H), 2.00 (m, 2H), 1.72 (m, 1H),1.60 (m, 2H), 0.81 (m, 6H). ¹³C (D₂O, 125 MHz) δ ppm 170.51, 58.78,53.69, 47.97, 45.36, 38.09, 24.34, 22.07, 21.51, 20.82. [α]_(D)=+13.1°(c=0.0019 in water), ES-MS 266 (M−1).

Preparation of3-{[(1R)-1-(aminocarbonyl)-2-methylpropyl]amino}1-propanesulfonic acid(Compound HD)

D-Valinamide hydrochloride (2.49 g, 14.7 mmol) was treated with asolution of K₂CO₃ (50 mL). The organic mixture was extracted with EtOAc(3×50 mL). The organic extracts were separated, combined, dried withNa₂SO₄, filtered, evaporated under reduced pressure and dried in vacuo.

To a solution of D-valinamide (1.76 g, 14.7 mmol) in acetonitrile (30mL) was slowly added 1,3-propanesultone (1.75 g, 14.4 mmol). Thesolution was stirred at reflux for 2 hours. The reaction mixture wascooled to room temperature. The solid was collected by filtration,washed with acetonitrile (2×25 mL). The solid product was dissolved inwater (75 mL). Dowex Marathon C resin (strongly acidic) was added to thesolution. The suspension was stirred for 15 minutes before the resin wasremoved by filtration. The filtrate was evaporated under reducedpressure. The solid material was suspended in acetone (50 mL), filteredand dried in vacuo, affording the title compound (1.57 g, 51%). ¹H NMR(D₂O, 500 MHz) δ ppm 3.80 (m, 1H), 3.19 (m, 2H), 3.00 (m, 2H), 2.25 (m,1H), 2.16 (m, 2H), 1.08 (d, 3H, J=6.8 Hz), 1.02 (d, 3H, J=6.8 Hz). ¹³C(D₂O, 125 MHz) δ ppm 169.94, 65.86, 48.10, 46.27, 29.54, 21.23, 17.99,17.02. [α]_(D)=−12.4° (c=0.0037 in water), ES-MS 237 (M−1).

Preparation of3-{[(1R)-1-carbamoyl-2-phenylethyl]amino}-1-propanesulfonic acid(Compound HE)

D-Phenylalaninamide hydrochloride (2.53 g, 12.6 mmol) was treated with asaturated solution of K₂CO₃ (50 mL). The mixture was extracted withEtOAc (3×50 mL). The organic layers were separated, combined, dried withNa₂SO₄, filtered and evaporated under reduced pressure.

To a solution of D-Phenylalaninamide (1.83 g, 11.1 mmol) in acetonitrile(20 mL) was added 1,3-propane sultone (1.29 g, 10.6 mmol). The solutionwas stirred at reflux for 2.5 hours. The reaction mixture was cooled toroom temperature. The solid was filtered and washed with acetonitrile(2×20 mL). The solid product was suspended in EtOH (75 mL). Thesuspension was stirred at reflux for 1 hours. The solid material wasfiltered, washed with acetone (1×25 mL) and dried in vacuo, affordingthe title compound (2.62 g, 89%). ¹H NMR (D₂O, 500 MHz) δ ppm 7.28 (m,3H), 7.19 (m, 2H), 4.05 (m, 1H), 3.19 (dd, 1H, J=5.3 Hz, 14.2 Hz), 3.04(m, 3H), 2.86 (t, 2H, J=5.8 Hz), 2.03 (m, 2H). ¹³C (D₂O, 125 MHz) δ ppm170.39, 133.73, 129.62, 129.26, 128.15, 61.57, 47.99, 45.57, 36.21,21.45. [α]_(D)=−20.7° (c=0.0038 in water), ES-MS 285 (M−1).

Preparation of3-({(1S)-1-[(benzyloxy)carbonyl]-2-methylbutyl}amino)-1-propanesulfonicacid (Compound HF)

L-Isoleucine benzylester p-tosylate (2.50 g, 6.4 mmol) was treated witha saturated solution of K₂CO₃ (30 mL). The mixture was extracted withEtOAc (3×30 mL). The organic extracts were separated, combined, driedwith Na₂SO₄, filtered and evaporated under reduced pressure.

To a solution of L-isoleucine benzylester (1.41 g, 6.4 mmol) inacetonitrile (12 mL) was added 1,3-propane sultone (706 mg, 5.8 mmol).The solution was stirred at reflux for 2 hours. The reaction mixture wascooled to room temperature. The solid was filtered and washed withacetone (2×20 mL). The solid material was dissolved in 50% EtOH/water(50 mL). Dowex Marathon C ion exchange resin (strongly acidic, 10 g) wasadded to the solution. The suspension was stirred for 15 minutes beforethe resin was removed by filtration. The filtrate was evaporated underreduced pressure and lyophilized affording the title compound (778 mg,39%). ¹H NMR (D₂O, 500 MHz) δ ppm 7.49 (m, 5H), 5.42 (d, 1H, J=11.7 Hz),5.31 (d, 1H, J=11.7 Hz), 3.24 (m, 2H), 2.98 (m, 2H), 2.13 (m, 3H), 1.46(m, 1H), 1.34 (m, 1H), 0.93 (m, 6H). ¹³C (D₂O, 125 MHz) δ ppm 168.53,134.69, 129.28, 129.22, 129.06, 68.86, 64.45, 48.12, 46.56, 36.21,25.97, 21.31, 13.73, 11.05. [α]_(D)=−1.5° (c=0.0031 in water), ES-MS 342(M−1).

Preparation of3-{[(1R)-1-(aminocarbonyl)-3-methylbutyl]amino}-1-propanesulfonic acid(Compound HG)

D-Leucinamide hydrochloride (1.0 g, 6.0 mmol) was treated with asaturated solution of K₂CO₃ (30 mL). The aqueous mixture was extractedwith EtOAc (3×30 mL). The organic extracts were separated, combined,dried with Na₂SO₄, filtered and evaporated under reduced pressure.

To a solution of D-Leucinamide (6.0 mmol) in acetonitrile (35 mL) wasadded 1,3-propanesultone (666 mg, 5.5 mmol). The solution was stirred atreflux for 2 hours. The reaction mixture was cooled to room temperature.The solid was filtered and washed with MeCN (2×20 mL). The solid wassuspended in EtOH (50 mL). The suspension was stirred at reflux for 1hour. The mixture was cooled to room temperature. The solid material wasfiltered, washed with acetone (1×20 mL) and dried in a vacuum oven (50°C.), affording the title compound (1.03 g, 74%): ¹H NMR (D₂O, 500 MHz) δppm 3.81 (m, 1H), 3.07 (m, 2H), 2.85 (t, 2H, J=7.3 Hz), 2.03 (m, 2H),1.68 (t, 1H, J=7.8 Hz), 1.58 (m, 2H), 0.83 (m, 6H). ¹³C (D₂O, 125 MHz) δppm 171.45, 59.39, 48.01, 45.41, 39.02, 24.24, 22.21, 21.47, 21.13.[α]_(D)=−13.7° (c=0.0019 in water), ES-MS 251 (M−1).

3-[(1-methylcyclopentyl)amino]-1-propanesulfonic acid (Compound FQ)

For the Ritter reaction, the flask was closed with a septum andconnected to a 20% NaOH scrubber. Sodium cyanide (powdered, 5.5 g, 112mmol) was added to acetic acid (30 mL) in one portion. The mixture wasstirred for 10 minutes at room temperature. A solution of sulfuric acid(16 mL) in acetic acid (15 mL) was added dropwise over a 20 minuteperiod. Then, a solution of 1-methyl-1-cyclopentanol (10 g, 99.8 mmol)in acetic acid (5 mL) was added dropwise over a 5 minute period. Themixture was stirred at room temperature for 22 hours then poured overice (approx. 100 g). The pH of the solution was adjusted to 9 with theaddition of 50% NaOH (about 135 g). The layers were separated and theaqueous layer was extracted with ether (1×40 mL). The combined organiclayers were washed with saturated sodium carbonate (1×10 mL) then driedover sodium sulfate. The ether was evaporated under reduced pressure toafford light brown oil (12.04 g, 94.7 mmol, 95%). The oil showed to be amixture of cis and trans formamide but what otherwise pure enough to beused as such. ¹H NMR (500 MHz, CDCl₃) δ [1.40 and 1.45 (s, 3H)],1.68-1.76 (m, 7H), 1.97-1.98 (m, 1H), [5.42 and 6.24 (br s, 1H)], [8.05(s) and 8.24 (d, J=12.2 Hz) for 1H]; ¹³C NMR (125 MHz, CDCl₃) δ 23.1,23.6, 25.5, 28.2, 39.5, 40.7, 60.7, 61.3, 160.7, 163.9

A solution of NaOH (25%, 80 mL) was added to the crude1-methyl-1-cyclopentylformamide (12.00 g, 94.7 mmol). The mixture washeated to reflux for 2.5 hours then cooled at room temperature. Somesodium chloride (20 g) was added to facilitate the phase separation. Thelayers were separated and the aqueous layer was extracted with toluene(1×15 mL). The combined organic layers were agitated. The addition ofisopropyl ether (2.5 mL, chloroform (1 g) and cyclohexane (6.5 g) didnot improved the seperation of the solution. The combined organic layerswere washed with brine (1×10 mL) then dried over sodium sulfate andfiltered. The filtrate was used as such in the next step. ¹H NMR (500MHz, CDCl₃) δ 1.22 (s, 3H), 1.47-1.75 (4 m, 9H): ¹³C NMR (125 MHz,CDCl₃) δ 24.1, 29.6, 42.2, 58.4

A solution of 1,3-propanesultone (9.4 g, 75 mmol) in 2-butanone (35 mL)was added dropwise to a the crude solution of1-methyl-1-cyclopentylamine (mixture of solvent from previous step).

The mixture was heated to reflux for 20 hours then was cooled to roomtemperature. The solid was collected by suction-filtration and rinsedwith acetone (2×10 mL). The solid was dried overnight at 45° C. in thevacuum oven. The title compound was obtained as a fine white solid(16.26 g, 73.47 mmol, 74% overall yield). ¹H NMR (500 MHz, D₂O) δ 1.31(s, 3H), 1.591.6-1-85 (m, 8H), 2.03-2.06 (m, 2H), 2.96 (t, J=6.8 Hz,2H), 3.15 (t, J=7.6 Hz, 2H); ¹³C NRM (125 MHz, D₂O) δ 22.1, 22.5, 23.6,36.5, 41.4, 48.1, 66.8.1; ES-MS 220 (M−H)

3-[(1-methylcyclohexyl)amino]-1-propanesulfonic acid (Compound FR)

For the Ritter reaction, the flask was closed with a septum andconnected to a 20% NaOH scrubber. Potassium cyanide (powdered, 3.3 g, 50mmol) was added in portions to acetic acid (13 mL). The mixture wasstirred for 10 minutes at room temperature. A solution of sulfuric acid(7 mL) in acetic acid (7 mL) was added drop-wise over a 10 minuteperiod. Then, a solution of 1-methyl-1-cyclohexanol (5 g, 43.8 mmol) inacetic acid (4 mL) was added dropwise over a 5 minute period. Themixture was stirred at room temperature for 22 hours then poured overice (approx. 50 g). The pH of the solution was adjusted to 9 with theaddition of 50% NaOH (about 70 g). The layers were separated and theaqueous layer was extracted with ether (2×20 mL). The combined organiclayers were washed with saturated sodium carbonate (1×10 mL) then driedover sodium sulfate. The ether was evaporated under reduced pressure toafford a clear yellow oil (6.56 g, quantitative). The oil showed to be amixture of cis and trans formamide but what otherwise pure enough to beused as such. ¹H NMR (500 MHz, CDCl₃) δ 1.33-1.53 (3m, 11H), 1.67 (br s,1H), 1.99 (m, 1H), [5.16 and 6.09 (br s, 1H)], [8.11 (s) and 8.25 (d,J=12.2 Hz) for 1H]; ¹³C NMR (125 MHz, CDCl₃) δ 23.1, 23.6, 25.5, 28.2,39.5, 40.7, 60.7, 61.3, 160.7, 163.9

A solution of NaOH (20%, 40 mL) was added to the crude1-methyl-1-cyclohexylformamide (43.8 mmol). The mixture was heated toreflux for 3 hours then cooled at room temperature. Some sodium chloride(7.5 g) was added to facilitate the phase separation. The layers wereseparated and the aqueous layer was extracted with MTBK (1×10 mL). Thecombined organic layers were washed with brine (1×5 mL) the dried oversodium sulfate and filtered. The filtrate was used as such in the nextstep. ¹H NMR (500 MHz, CDCl₃) δ 1.08 (s, 3H), 1.33-1.51 (m, 10H); ¹³CNMR (125 MHz, CDCl₃) δ 22.8, 25.8, 29.6, 40.8, 48.6

A solution of 1,3-propanesultone (5.00 g, 40 mmol) in toluene (10 mL)was added dropwise to a the crude solution of 1-methyl-1-cyclohexylaminein MTBK (total volume 30 mL). The mixture was heated to reflux for 18hours then cooled to room temperature. The solid was collected bysuction filtration, rinsed with acetone (2×8 mL). The solid was driedovernight at 45° C. in the vacuum oven. The title compound was obtainedas a fine white solid (9.22 g). However, the proton NMR and the ES-MSwere not clean. The solid was suspended in methanol (45 mL) and thesuspension was warmed to reflux. Water (12 mL) was added dropwise untila clear yellow solution was obtained. The mixture was slowly cool toroom temperature with stirring. The solid was collected bysuction-filtration, rinsed with methanol (2×5 mL). Another crop wascollected from the filtrate. Both crops were dried overnight at 45° C.in the vacuum oven. The title compound was obtained as a fine whitesolid (6.82 g, 29.0 mmol, 66% overall yield). Both crops were identicaland were mixed for submitting the compound. ¹H NMR (500 MHz, D₂O) δ1.04-1.11 (m, 1H), 1.19 (s, 3H), 1.31 (q, J=12.2 Hz, 2H), 1.40 (qt,J=12.2 Hz, 2H), 1.46-1.62 (m, 2H), 1.63 (br d, J=11.7 Hz, 2H), 1.94 (q,J=7.3 Hz, 2H), 2.86 (t, J=7.1 Hz, 2H), 3.03 (t, J=7.6 Hz, 2H); ¹³C NMR(125 MHz, D₂O) δ 19.1, 21.5, 22.0, 24.6, 34.1, 39.1, 48.2, 60.2; ES-MS236 (M+H).

3-[(1-methylcycloheptyl)amino]-1-propanesulfonic acid (Compound FS)

For the Ritter reaction, the flask was closed with a septum andconnected to a 20% NaOH scrubber. Potassium cyanide (powdered, 2.8 g, 43mmol) was added in portions to acetic acid (10 mL). The mixture wasstirred for 10 minutes at room temperature. A solution of sulfuric acid(7 mL) in acetic acid (7 mL) was added drop-wise over a 20 minuteperiod. Then, the 1-methyl-1-cycloheptanol (5 g, 39.0 mmol) was addeddrop-wise over 5 minutes. The mixture was stirred at room temperaturefor 22 h then cooled to 0° C. with a ice/water bath. The pH of thesolution was adjusted to 9 with the addition of 50% NaOH (about 70 g).The layers were separated and the aqueous layer was extracted with ether(1×20 mL). The combined organic layers were washed with saturated sodiumcarbonate (1×5 mL) then dried over sodium sulfate. The ether wasevaporated under reduced pressure to afford a clear yellow oil (5.71 g,94%). The oil showed to be a mixture of cis and trans formamide but whatotherwise pure enough to be used as such. ¹H NMR (500 MHz, CDCl₃) δ 1.34(s, 1.5H), 1.43 (s, 1.5H), 1.49-1.60 (m, 8H), 1.96-2.00 (m, 1H), [5.28and 5.95 (br s, 1H)], [8.06 (s) and 8.28 (d, J=12.2 Hz) for 1H]; ¹³C NMR(125 MHz, CDCl₃) δ 22.2, 22.4, 27.7, 29.3, 29.4, 30.7, 40.5, 42.5, 56.0,57.4, 160.5, 163.3

A solution of NaOH (25%, 40 mL) was added to the crude1-methyl-1-cycloheptylformamide (5.7 g). The mixture was heated toreflux for 3 hours then cooled at room temperature. Some sodium chloride(7.5 g) was added to facilitate the phase separation. The layers wereseparated and the aqueous layer was extracted with MTBK (1×10 mL). Thecombined organic layers were washed with brine (1×5 mL) the dried oversodium sulfate and filtered. The filtrate was used as such in the nextstep. ¹H NMR (500 MHz, CD₃OD) δ 1.10 (s, 3H), 1.40-1.48 (m, 2H),1.5-1.65 (m, 10H); ¹³C NMR (125 MHz, CD₃OD) δ 24.0, 31.2, 31.4, 44.4,53.6

A solution of 1,3-propanesultone (4.3 g, 35 mmol) in toluene (10 mL) wasadded dropwise to a the crude solution of 1-methyl-1-cycloheptylamine inMTBK (total volume 30 mL). The mixture was heated to reflux for 18 hoursthen was cooled to room temperature. The solid was collected by suctionfiltration, rinsed with acetone (2×5 mL). The solid was dried overnightat 45° C. in the vacuum oven. The title compound was obtained as a finewhite solid (7.77 g, 31.2 mmol, 80% overall yield). ¹H NMR (500 MHz,D₂O) δ 1.27 (s, 3H), 1.40-1.60 (m, 8H), 1.71-1.81 (m, 4H), 2.00-2.06 (m,2H), 2.95 (t, J=6.3 Hz, 2H), 3.13 (t, J=7.1 Hz, 2H); ¹³C NMR (125 MHz,D₂O) δ 22.0, 22.1, 23.3, 29.5, 37.1, 40.0, 48.3, 64.0; ES-MS 250(M+H)

Preparation of3-{[(1R)-1-(benzyloxycarbonyl)-3-methylbutyl]amino}-1-propanesulfonicacid (Compound HI)

D-Leucine benzylester p-tosylate (2.5 g, 6.3 mmol) was treated with anaqueous solution of K₂CO₃ (30 mL). The mixture was extracted with EtOAc(3×30 mL). The organic extracts were separated, combined, dried withNa₂SO₄, filtered, evaporated under reduced pressure and dried in vacuo.

To a solution of D-Leucine benzylester (6.3 mmol) in acetonitrile (9 mL)and MeOH (3 mL) was added 1,3-propane sultone (691 mg, 5.7 mmol). Thesolution was stirred at reflux for 2.5 hours. The reaction mixture wascooled to room temperature. The solid material was filtered and washedwith aconitrile (2×20 mL). The solid was dissolved in 20% water/EtOH (75mL). Dowex Marathon C ion exchange resin (strongly acidic) was added tothe solution. The suspension was stirred for 15 minutes before the resinwas removed by filtration. The filtrate was evaporated under reducedpressure and dried in vacuo, affording the title compound (960 mg, 49%).¹H NMR (D₂O, 500 MHz) δ ppm 7.52 (m, 5H), 5.41 (d, 1H, J=12.2 Hz), 5.35(d, 1H, J=12.2 Hz), 4.16 (m, 1H), 3.22 (m, 2H), 2.97 (t, 2H, J=6.8 Hz),2.16 (m, 2H), 1.88 (m, 1H), 1.79 (m, 1H), 1.76 (m, 1H), 0.94 (d, 6H,J=3.9 Hz). ¹³C (DMSO, 125 MHz) δ ppm 169.60, 135.62, 129.24, 129.21,129.11, 68.08, 58.09, 49.87, 46.48, 24.77, 23.50, 22.50, 22.04.[α]_(D)=−2.1° (c=0.00095 in water), ES-MS 344 (M+1).

Preparation of 3-[(5-hydroxy-1,5-dimethylhexyl)amino]-1-propanesulfonicacid (Compound HJ)

To a solution of 6-amino-2-methyl-2-heptanol (2.5 g, 17.2 mmol) inacetonitrile (22 mL) was added 1,3-propane sultone (2.0 g, 16.4 mmol).The solution was stirred at reflux for 2 hours. The reaction mixture wascooled to room temperature. The solid material was filtered and washedwith acetonitrile (2×20 mL). The solid was dissolved in 20% MeOH (75mL). Dowex Marathon C ion exchange resin (strongly acidic) was added tothe solution. The suspension was stirred for 15 minutes before the resinwas removed by filtration. The filtrate was evaporated under reducedpressure. The solid was suspended in acetone (150 mL), and then thesolid material was filtered and dried in vacuo, affording the titlecompound (3.08 g, 70%). ¹H NMR (D₂O, 500 MHz) δ ppm 3.19 (m, 1H), 3.08(m, 2H), 2.88 (t, 2H, J=7.3 Hz), 1.99 (m, 2H), 1.60 (m, 2H), 1.36 (m,4H), 1.18 (d, 3H, J=6.8 Hz), 1.07 (s, 6H). ¹³C (D₂O, 125 MHz) δ ppm71.63, 54.73, 48.08, 43.46, 42.27, 32.97, 27.78, 27.73, 21.64, 19.67,15.43. ES-MS 268 (M+1).

Preparation of3-{[(1R)-2-methoxy-1-methyl-2-oxoethyl]amino}-1-propanesulfonic acid(Compound HK)

D-Alanine methylester hydrochloride (3.0 g, 21.5 mmol) was treated witha aqueous solution of K₂CO₃ (50 mL). The mixture was extracted withEtOAc (3×50 mL). The organic extracts were separated, combined, driedwith Na₂SO₄, filtered and evaporated under reduced pressure.

To a solution of D-Alanine methylester (1.33 g, 12.9 mmol) inacetonitrile (15 mL was added 1,3-propane sultone (1.42 g, 11.7 mmol).The solution was stirred at reflux for 2 hours. The reaction mixture wascooled to room temperature. The solid material was filtered and washedwith acetonitrile (2×15 mL). The solid was dissolved in water (30 mL).Dowex Marathon C ion exchange resin (strongly acidic) was added to thesolution. The suspension was stirred for 15 minutes before the resin wasremoved by filtration. The filtrate was evaporated under reducedpressure and dried in vacuo, affording the title compound (1.52 g, 42%).¹H NMR (D₂O, 500 MHz) δ ppm 4.07 (m, 1H), 3.72 (s, 3H), 3.14 (m, 2H),2.89 (t, 2H, J=7.3 Hz), 2.03 (m, 2H), 1.46 (dd, 3H, J=1.95 Hz, 7.3 Hz).¹³C (DMSO, 125 MHz) δ ppm 170.74, 55.62, 53.82, 47.96, 44.76, 21.53,14.03. [α]_(D)=+1.4° (c=0.0088 in water), ES-MS 224 (M−1).

Preparation of 4-(1,2,3,4-tetrahydro-1-naphthylamino)-2-butanesulfonicacid (Compound JF)

To a solution of 1,2,3,4-tetrahydro-1-naphthylamine (2.01 g, 13.6 mmol)in 2-butanone (15 mL) was added 2,4-butane sultone (1.95 g, 14.3 mmol).The solution was stirred at for 2 hours. The reaction was cooled to roomtemperature. The solid was collected by filtration, washed with acetone(2×25 mL) and dried in vacuo. ¹H NMR (DMSO, 500 MHz) δ ppm 8.56 (s(broad), 1H), 7.49 (dd, 1H, J=8.0 Hz, 11.9 Hz), 7.29 (m, 1H), 7.26 (m,1H), 7.19 (d, 1H, J=8.0 Hz), 4.40 (d, 1H, J=13.7 Hz), 3.14 (m, 2H), 2.75(m, 3H), 1.96 (m, 5H), 1.40 (m, 1H), 1.23 (m, 3H). ¹³C (DMSO, 125 MHz) δppm 138.81, 131.81, 130.40, 130.28, 130.16, 129.41, 126.76, 126.73,54.97, 54.58, 54.08, 44.18, 43.23, 29.50, 28.84, 24.84, 24.76, 18.23,18.20, 17.79, 17.01. ES-MS 284 (M+1).

Preparation of 4-(octylamino)-2-butanesulfonic acid (Compound JG)

To a solution of octylamine (2.00 g, 15.5 mmol) in 2-butanone (17 mL)was added 2,4-butane sultone (2.21 g, 16.2 mmol). The solution wasstirred at reflux for 2 hours. The reaction was cooled to roomtemperature. The solid was collected by filtration, washed with acetone(2×25 mL) and dried in vacuo. It was suspended in a solution of 25%EtOH/acetone (50 mL). The suspension was stirred for 5 minutes. Thesolid was collected by filtration, washed with acetone (2×25 mL) anddried in vacuo. ¹H NMR (DMSO, 500 MHz) δ ppm 8.45 (s (broad), 1H), 3.01(m, 1H), 2.84 (m, 2H), 2.58 (m, 1H), 1.92 (m, 1H), 1.75 (m, 1H), 1.51(m, 2H), 1.10 (d, 1H, J=6.8 Hz), 0.85 (t, 3H, J=6.8 Hz). ¹³C (DMSO, 125MHz) δ ppm 53.05, 47.27, 46.15, 31.83, 29.42, 29.13, 26.51, 26.27,22.75, 17.19, 14.64. ES-MS 266 (M+1).

Preparation of 4-(adamantyl)amino-2-butanesulfonic acid (Compound JH)

1-adamantaneamine hydrochloride (2.51 g, 13.3 mmol) was treated with 1NNaOH (20 mL) and CH₂Cl₂ (3×20 mL). The organic extracts were combined,dried with Na₂SO₄, filtered, evaporated under reduced pressure and driedin vacuo.

To a solution of 2-adamantanamine (1.99 g, 13.1 mmol) in acetonitrile(15 mL) was added 2,4-butane sultone (1.87 g, 13.8 mmol). The solutionwas stirred at reflux for 2 hours. The reaction was cooled to roomtemperature. The solid was collected by filtration, washed withacetonitrile (3×25 mL) and dried in vacuo. ¹H NMR (DMSO, 500 MHz) δ ppm8.53 (s (broad), 1H), 3.33 (m, 2H), 2.61 (m, 1H), 2.10 (s, 3H), 1.93 (m,1H), 1.77 (m, 7H), 1.61 (m, 6H), 1.12 (d, 1H, J=6.8 Hz). ¹³C (DMSO, 125MHz) δ ppm 56.20, 53.34, 35.85, 29.75, 29.04, 17.206. ES-MS 288 (M+1).

Preparation of 4-(2-adamantyl)amino-2-butanesulfonic acid (Compound JI)

The 2-adamantanamine hydrochloride (2.50 g, 13.3 mmol) was treated with1N NaOH (2.0 mL) and CH₂Cl₂ (3×20 mL). The organic extracts werecombined, dried with Na₂SO₄, filtered, evaporated under reduced pressureand dried in vacuo.

To a solution of 1-adamantanamine (1.99 g, 13.1 mmol) in acetonitrile(15 mL) was added 2,4-butane sultone (1.87 g, 13.8 mmol). The solutionwas stirred at reflux for 2 hours. The reaction was cooled to roomtemperature. The solid was collected by filtration, washed withacetonitrile (2×25 mL) and dried in vacuo. ¹H NMR (DMSO, 500 MHz) δ ppm3.20 (m, 1H), 3.05 (m, 2H), 2.67 (m, 1H), 2.07 (m, 2H), 2.00 (m, 1H),1.95 (m, 4H), 1.82 (m, 4H), 1.69 (m, 4H), 1.55 (m, 4H), 1.12 (d, 1H,J=8.0 Hz). ¹³C (DMSO, 125 MHz) δ ppm 62.27, 53.91, 44.33, 37.30, 36.82,36.77, 30.30, 30.20, 29.57, 29.50, 28.95, 17.12, 26.85, 17.44. ES-MS 288(M+1).

Preparation of 4-(bicyclo[2.2.1]hept-2-ylamino)-2-butanesulfonic acid(Compound JJ)

To a solution of exo-2-aminonorbornane (1.0 g, 9.0 mmol) intetrahydrofuran (THF, 10 mL) was added 2,4-butane sultone (1.28 g, 9.3mmol). The solution was stirred at reflux for 3 hours. The reaction wascooled to room temperature. The solid was collected by filtration,washed with THF (2×20 mL) and dried in vacuo. ¹H NMR (DMSO, 500 MHz) δppm 8.43 (s (broad), 1H), 2.96 (m, 3H), 2.62 (m, 1H), 2.38 (m, 1H),2.28, (m, 1H), 1.91 (m, 1H), 1.82 (m, 1H), 1.61 (m, 1H), 1.54 (m, 2H),1.42 (m, 2H), 1.12 (m, 6H). ¹³C (DMSO, 125 MHz) δ ppm 60.92, 60.79,53.61, 53.21, 44.55, 44.36, 39.80, 39.55, 36.27, 36.15, 36.11, 35.98,35.19, 35.13, 29.62, 29.43, 28.07, 26.88, 17.56, 14.11. ES-MS 248 (M+1).

Preparation of 4-(azoniabicyclo[2.2.2]oct-2-ylamino)-2-butanesulfonate(Compound JK)

Quinuclidine hydrochloride (2.50 g, 16.9 mmol) was treated with 1N NaOH(20 mL) and CH₂Cl₂ (4×20 mL). The organic extracts were combined, driedwith Na₂SO₄, filtered, evaporated under reduced pressure and dried invacuo.

To a solution of quinuclidine (900 g, 8.2 mmol) in tetrahydrofuran (THF,18 mL) and MeOH (0.5 mL) was added 2,4-butane sultone (1.16 g, 8.6mmol). The solution was stirred at reflux overnight. The reaction wascooled to room temperature. The solid was collected by filtration,washed with THF (2×25 mL) and dried in vacuo. ¹H NMR (DMSO, 500 MHz) δppm 3.40 (m, 7H), 3.20 (td, 1H, J=3.9 Hz, 12.7 Hz), 2.38 (m, 1H), 2.01(m, 2H), 1.83, (m, 6H), 1.70 (m, 1H), 1.10 (d, 3H, J=12.7 Hz). ¹³C(DMSO, 125 MHz) δ ppm 62.63, 54.23, 52.18, 25.67, 24.07, 19.78, 17.04.ES-MS 208 (M+1).

Preparation of 4-[(dl)-1-hydroxy-2-pentyl]amino-2-butane sulfonic acid(Compound JL)

To a solution of DL-2-aminopentanol (1.0 g, 9.7 mmol) in tetrahydrofuran(11 mL) was added 2,4-butane sultone (1.45 g, 10.2 mmol). The solutionwas stirred at reflux for 4 hours. The reaction was cooled to roomtemperature. The supernatant was removed and the solid was dried invacuo. The product was suspended in 2-propanol (100 mL) and the mixturewas stirred for 5 minutes. The white solid was filtered, washed with2-propanol and dried in vacuo. ¹H NMR (D₂O, 500 MHz) δ ppm 3.74 (d, 1H,J=12.7 Hz), 3.59 (dd, 1H, J=5.4 Hz, 12.9 Hz), 3.13 (m, 3H), 2.89 (m,1H), 2.07 (m, 1H), 1.82, (m, 1H), 1.52 (m, 2H), 1.28 (m, 2H), 1.18 (d,3H, J=6.8 Hz). ¹³C (D₂O, 125 MHz) δ ppm 59.43, 58.80, 53.47, 42.88,29.30, 28.43, 18.40, 14.99, 13.23. ES-MS 240 (M+1).

Preparation of 4-(nonylamino)-2-butanesulfonic acid (Compound JN)

To a solution of nonylamine (2.00 g, 14.0 mmol) in tetrahydrofuran (THF,15 mL) was added 2,4-butane sultone (2.08 g, 14.7 mmol). The solutionstirred at reflux for 5 hours. The reaction was cooled to roomtemperature. The solid was collected by filtration, washed with THF(2×25 mL) and dried in vacuo.

The product (1.10 g, 3.9 mmol) was dissolved with heating in a solutionof EtOH (20 mL), water (600 uL) and NaOH (163 mg, 4.1 mmol). After a fewminutes a white solid precipitated. The solid was collected byfiltration, washed with acetone (2×25 mL) and dried in vacuo. ¹H NMR(D₂O, 500 MHz) δ ppm 2.70 (m, 1H), 2.52 (m, 2H), 2.37 (m, 1H), 1.95 (m,1H), 1.46 (m, 1H), 1.34 (m, 1H), 1.14 (m, 17H), 0.70 (t, 3H, J=6.8 Hz).¹³C (D₂O, 125 MHz) δ ppm 54.31, 52.90, 50.44, 31.31, 28.80, 28.74,28.57, 27.31, 26.95, 22.20, 14.60, 13.57. ES-MS 302 (M+1).

Preparation of 4-(dimethylamino)-2-butanesulfonic acid (Compound JO)

2,4-butanesultone (1.27 g, 8.9 mmol) was added to an ice-chilledsolution of dimethylamine (40% w/w in water). The solution was stirredat 0° C. for 4 hours. The solvent was evaporated in vacuo until completedryness. The solid was washed with acetone (50 mL), collected byfiltration and dried in vacuo. ¹H NMR (D₂O, 500 MHz) δ ppm 3.18 (t, 2H,J=8.1 Hz), 2.85 (m, 1H), 2.76 (s, 6H), 2.09 (m, 1H), 1.81 (m, 1H), 1.17(d, 3H, J=7.3 Hz). ¹³C (D₂O, 125 MHz) δ ppm 55.65, 53.05, 42.88, 26.72,14.81. ES-MS182a (M+1).

Preparation of 4-(benzylamino)-2-butanesulfonic acid, sodium salt(Compound JP)

To a solution of benzylamine (1.50 g, 14.0 mmol) in tetrahydrofuran(THF, 18 mL) was added 2,4-butane sultone (1.98 g, 14.6 mmol). Thesolution was stirred at reflux for 2 hours. The reaction was cooled toroom temperature. The solid was collected by filtration, washed with THF(2×25 mL) and dried in vacuo.

The product (2.55 g, 10.5 mmol) was dissolved with heating in a solutionof EtOH (25 mL), water (1.6 mL) and NaOH (440 mg, 11.0 mmol). Diethylether (150 mL) was added to the filtrate. The solid was filtered anddried in vacuo. Yield: 27%. ¹H NMR (DMSO, 500 MHz) δ ppm 7.29 (m, 4H),7.20 (m, 1H), 3.67 (m, 2H), 2.56 (m, 1H), 2.45 (m, 2H), 1.98 (m, 1H),1.36 (m, 1H), 1.04 (d, 3H, J=6.8 Hz). ¹³C (DMSO, 125 MHz) δ ppm 141.49,128.73, 128.61, 127.16, 53.49, 52.88, 47.31, 32.67, 16.53. ES-MS 266(M+1).

Preparation of 4-(ethylamino)-2-butanesulfonic acid, sodium salt(Compound JQ)

A solution of 2,4-butanesultone (1.33 g, 9.3 mmol) in tetrahydrofuran(THF, 3.0 mL) was added via syringe pump over a 2 h period to ethylamine(70% w/w in water, 12.0 mL, 186.0 mmol) at 5° C. The solution wasstirred at 5° C. for an additional 2 hours. The solvent was coevaporatedwith EtOH (3×25 mL). The solid was suspended in acetone (25 mL). Thesuspension was stirred for 5 minutes the solid was filtered, washed withacetone (2×25 mL) and dried in vacuo. Yield: 70%. ¹H NMR (D₂O, 500 MHz)δ ppm 3.06 (t, 2H, J=8.1 Hz), 2.97 (m, 2H), 2.87 (m, 2H), 2.06 (m, 1H),1.77 (m, 1H), 1.18 (d, 3H, J=7.3 Hz), 1.14 (t, 3H, J=7.3 Hz). ¹³C (D₂O,125 MHz) δ ppm 53.16, 44.91, 43.03, 28.20, 14.76, 10.66. ES-MS 182(M+1).

Preparation of 4-(tert-butylamino)-1-butanesulfonic acid (Compound LD)

To a solution of tert-butylamine (1.0 mL, 9.5 mmol) in tetrahydrofuran(4 mL) was added 1,4-butane sultone (1.36 g, 10.0 mmol) at roomtemperature. The solution was stirred at reflux for 2 hours. Thereaction was cooled to room temperature. The solid was collected byfiltration, washed with acetone (2×20 mL) and dried in vacuo. Yield: 690mg (34%). ¹H NMR (D₂O, 500 MHz) δ ppm 2.92 (t, 2H, J=7.1 Hz), 2.82 (t,2H, J=7.1 Hz), 1.68 (m, 4H), 1.22 (s, 9H). ¹³C (D₂O, 125 MHz) δ ppm57.07, 50.30, 40.95, 25.28, 24.96, 21.62. ES-MS 210 (M−1).

Preparation of 4-amino-2-butanesulfonic acid (Compound JR)

A solution of 2,4-butanesultone (1.0 g, 7 mmol) in tetrahydrofuran (THF,4.0 mL) was added via syringe pump over a 4 h period to ammoniumhydroxide (28-30% NH₃, 43 mL, 350 mmol) at 5° C. The solution wasstirred at 5° C. for an additional 30 minutes. The solvent wascoevaporated with EtOH (3×25 mL). The solid was dried in vacuo. Yield:94%. ¹H NMR (D₂O, 500 MHz) δ ppm 3.05 (m, 2H), 2.90 (m, 1H), 2.05 (m,1H), 2.06 (m, 1H), 1.77 (m, 1H), 1.18 (d, 3H, J=6.8 Hz), 1.14 (t, 3H,J=7.3 Hz). ¹³C (DMSO, 125 MHz) δ ppm 52.75, 38.20, 30.87, 17.27. ES-MS154 (M+1).

Preparation of 4-piperidin-1-yl-2-butanesulfonic acid (Compound JS)

To a solution of piperidine (1.50 g, 17.6 mmol) in tetrahydrofuran (THF,20 mL) was added 2,4-butanesultone (2.50 g, 18.5 mmol). The solution wasstirred at reflux for 3 hours. The reaction was cooled to roomtemperature. The solid was collected by filtration, washed with THF(2×20 mL) and dried in vacuo.

The product (3.53 g, 15.9 mmol) was dissolved with heating in a solutionof EtOH (30 mL), water (1.3 mL) and NaOH (670 mg, 16.7 mmol). Thesolution was poured in a large excess of Et₂O (500 mL). The solid wasfiltered, washed with Et₂O (1×25 mL) and acetone (1×20 mL) and dried invacuo. Yield: 64%. ¹H NMR (D₂O, 500 MHz) δ ppm 2.72 (m, 1H), 2.33 (m,6H), 1.97 (m, 1H), 1.48 (m, 1H), 1.43 (m, 4H), 1.31 (m, 1H), 1.13 (d,3H, J=6.8 Hz). ¹³C (D₂O, 125 MHz) δ ppm 55.78, 54.42, 53.61, 27.73,24.92, 23.59, 14.61. ES-MS 244 (M+1).

Preparation of 4-(ethylamino)-1-butanesulfonic acid (Compound LE)

A solution of 1,4-butanesultone (2.66 g, 18.6 mmol) in tetrahydrofuran(total volume: 4 mL) was added via syringe pump over a 4 hour period toethylamine (70% w/w in water, 24 mL, 372 mmol) at 5° C. The solution wasstirred at 5° C. for an additional 3 hours before it was warm up to roomtemperature. The reaction was stirred in these conditions overnight. Thesolvent was coevaporated with EtOH (1×25 mL). The solid was suspended in50% acetone/EtOH (50 mL). The suspension was stirred for 5 minutes, thesolid was filtered and dried in vacuo. Yield: 75%. ¹H NMR (D₂O, 500 MHz)δ ppm 2.95 (m, 4H), 2.82 (m, 2H), 1.68 (m, 4H), 1.13 (t, 3H, J=7.3 Hz),1.14 (t, 3H, J=7.3 Hz). ¹³C (D₂O, 125 MHz) δ ppm 50.27, 46.68, 42.99,24.66, 21.48, 10.64. ES-MS182 (M+1).

Preparation of 4-(azoniabicyclo[2.2.2]oct-2-ylamino)-1-butanesulfonate(Compound LF)

To a solution of quinuclidine (1.5 g, 13.5 mmol) in tetrahydrofuran(THF, 15 mL) was added 1,4-butanesultone (2.0 g, 14.4 mmol) at roomtemperature. The solution was stirred at reflux for 2 hours. Thereaction was cooled to room temperature. The solid was collected byfiltration, washed with THF (2×25 mL) and dried in vacuo. ¹H NMR (D₂O,500 MHz) δ ppm 3.26 (m, 6H), 3.02 (m, 2H), 2.82 (t, 2H, J=17.3 Hz), 2.04(m, 1H), 1.84, (m, 6H), 1.75 (m, 2H), 1.64 (m, 2H). ¹³C (D₂O, 125 MHz) δppm 63.68, 54.81, 50.14, 23.51, 21.45, 20.58, 19.19. ES-MS 248 (M+1).

Preparation of 3-(dimethylamino)-2-hydroxy-1-propane sulfonic acid,sodium salt (Compound JT)

A solution of 3-chloro-2-hydroxy-1-propanesulfonic acid, sodium salt (10g, 48.3 mmol) in water (40 mL total volume) was added via a syringe pumpover 4 hours to a cold (2.8-3.1° C.) solution of dimethylamine (40% wtin water, 300 mL) with stirring. The mixture was slowly warmed to roomtemperature overnight. The mixture was then coevaporated with absoluteethanol (20 mL) and concentrated to dryness. The solid was driedovernight at 60° C. in the vacuum oven. The solid was suspended inethanol (40 mL) stirred at reflux for 2 hours. The suspension was cooledto 5° C. and the solid was collected by suction-filtration,aspirator-dried 5 minutes, then dried for the weekend at 60° C. in thevacuum oven (wet cake: 13.74 g). The desired material was obtained as anoff-white solid (11.65 g, quantitative).

Preparation of 4-Dimethylamino-1-butanesulfonic acid (Compound LH)

A solution of 1,4-butane sultone (7.5 mL, 73.6 mmol) in 1,4-dioxane(total volume: 10 mL) was added over 4 hours via a syringe pump to acold (4.3° C.) solution of dimethylamine (40% wt in water, 275 mL). Themixture was stirred for 3 hours at 4° C. after the end of the addition,then overnight at room temperature. The mixture was concentrated todryness. The solid was suspended in absolute ethanol (50 mL) and themixture was heated to reflux for 90 minutes. The suspension was cooledto 5° C. and the solid was collected by suction-filtration, rinsed withethanol (2×10 mL). The solid was dried for 18 h at 60° C. in the vacuumoven. The desired material was obtained as a fine white powder 13.21 g,72.9 mmol, 99% yield. The ¹H and ¹³C NMR and MS were consistent with thestructure.

Preparation of 3-(ethylamino)-2-hydroxy-1-propanesulfonic acid (CompoundJU)

A solution of 3-chloro-2-hydroxy-1-propanesulfonic acid sodium salt (10g, 50.9 mmol) in water (total volume: 40 mL) was added over 5 hours, viaa syringe pump, to a cold (4.7° C.) solution of ethyl amine in water.The mixture was stirred for an additional 2 hours at 4.7° C. then for 18hours at room temperature. NMR: quantitative yield. The mixture wasconcentrated. A solid could not be obtained: the sodium salt was toohygroscopic. The solution was treated with Amberlite IR-120 Plus, acidform, ion-exchange resin to give the free acid. It was still toohygroscopic to be obtained as a solid form. Submitted as a solution:d=1.314 g/mL, 62.5% w/w of the free acid in water. The ¹H and ¹³C NMRand MS were consistent with the structure.

Preparation of 3-(tert-butylamino)-2-hydroxy-1-propanesulfonic acid(Compound JV)

A solution of 3-chloro-2-hydroxy-1-propanesulfonic acid, sodium salt (15g, 25 mmol) in water (12 mL total volume) was added over 5 minutes to amixture of tert-butylamine (12.5 mL), water (6 mL) and methanol (3 mL).The mixture was heated at 35° C. for 1 h, 40° C. for 1 h, 45° C. for 1.5hours. The mixture was the concentrated to a thick oil. The crudereaction mixture was passed over a column of Dowex 50×8 (125 g). Thefractions containing the product were concentrated to dryness. The solidwas dried overnight at 60° C. in the vacuum oven. The solid wasrecrystallized in a mixture of methanol (25 mL) and water (7 mL). Themixture was cooled slowly to room temperature, then to 5° C. The solidwas collected by suction-filtration, rinsed with ethanol (1×10 mL). Thesolid was then dried for 18 hours at 60° C. in the vacuum oven. Thedesired material was obtained as an off-white solid (3.11 g, 14.7 mmol,59%). The ¹H and ¹³C NMR and MS were consistent with the structure.

Preparation of 1-(N-octylamino)-2-hydroxy-1-propanesulfonic acid(Compound JW)

A solution of 3-chloro-2-hydroxy-1-propanesulfonic acid, sodium salt (4g, 20 mmol) in water (17.5 mL total volume) was added over 2 hours to amixture of octylamine (8 mL), water (20 mL) and 1,4-dioxane (11 mL) at70-75° C. The mixture was stirred at this temperature for another 2hours after the end of the addition. The 1,4-dioxane was removed underreduced pressure and the mixture was diluted with water (10 mL). Themixture was extracted with 40% ethyl acetate/hexane (3×40 mL). Theaqueous layer was concentrated then the mixture was passed over a columnof Dowex 50×8 (125 g). The fractions containing the pure product wereconcentrated to a thick oil then freeze-dried. The desired material wasobtained as a white fluffy solid (150 mg, 0.56 mmol, 3%). The ¹H and ¹³CNMR and MS were consistent with the structure.

Preparation of 1-(3-sulfo-2-hydroxypropyl) quinuclidinium, inner salt(Compound JX)

A solution of 3-chloro-2-hydroxy-1-propanesulfonic acid, sodium salt (2g, 10 mmol) in water (12 mL total volume) was added over 1 hour to amixture of quinuclidine (1.63 g, 4.7 mmol), water (10 mL) and1,4-dioxane (10 mL) at 80° C. The mixture was stirred at thistemperature for another 2 hours after the end of the addition. Thereaction mixture was concentrated then the mixture was passed over acolumn of Dowex 50×8 (125 g). The fractions containing the pure productwere concentrated to a white solid. The solid was dried for 18 hours at60° C. in the vacuum oven. The desired material was obtained as a whitesolid (1.92 g, 7.7 mmol, 77%). The ¹H and ¹³C NMR and MS were consistentwith the structure.

Preparation of 1-(N-benzylamino)-2-hydroxy-1-propanesulfonic acid,sodium salt (Compound JY)

A solution of 3-chloro-2-hydroxy-1-propanesulfonic acid, sodium salt (4g, 20 mmol) in water (12.5 mL total volume) was added over 2 hours to amixture of benzylamine (4.28 g, 40 mmol), water (10 mL) and 1,4-dioxane(5 mL) at 80° C. The mixture was stirred at this temperature for another2.5 hours after the end of the addition. The reaction mixture wasextracted with chloroform (2×40 mL). It was then concentrated todryness. The crude solid was recrystallized in a mixture of ethanol (30mL) and water (4 mL). The mixture was left to cool to room temperaturefor the night. The solid was collected by suction-filtration, rinsedwith ethanol (10 mL) and dried in the vacuum oven at 60° C. The desiredmaterial was obtained as a white solid (2.67 g, 10 mmol, 50%). The ¹Hand ¹³C NMR and MS were consistent with the structure.

Preparation of2-hydroxy-3-(1,2,3,4-tetrahydronaphthalen-1-ylamino)propane-1-sulfonicacid (Compound JZ)

A solution of 3-chloro-2-hydroxy-1-propanesulfonic acid, sodium salt (2g, 10 mmol) in water (9.75 mL total volume) was added over 8 hours to amixture of 1,2,3,4-tetrahydro-1-nahtylamine (2 g, 13.6 mmol), water (10mL) and 1,4-dioxane (4 mL) at 40° C. The mixture was stirred at thistemperature for another 18 hours after the end of the addition. Thereaction was not completed. The mixture was heated for 2 hours atreflux. The mixture was diluted with water (10 mL) and 50% w/w NaOH(0.25 mL) was added. The reaction mixture was extracted with chloroform(2×25 mL). It was then concentrated to a thick oil. The solution wasapplied on a Dowex 50 W 8 column (100 g). The fractions containing theproduct were concentrated, treated with activated charcoal (no effect)and freeze-dried. The desired material was obtained as a glassy solid(0.85 g, 3 mmol, 30%). The ¹H and ¹³C NMR and MS were consistent withthe structure.

Preparation of 2-hydroxy-3-piperidin-1-ylpropane-1-sulfonic acid(Compound KA)

A solution of 3-chloro-2-hydroxy-1-propanesulfonic acid, sodium salt (4g, 20 mmol) in water (13.35 mL total volume) was added over 5 hours to asolution of piperidine (8 mL g, 80 mmol), in water (15 mL) at 70° C. Themixture was stirred at 80° C. for 2 hours. The reaction was completed.The mixture was stirred at room temperature for the night. The mixturewas diluted with water (10 mL) and was extracted with chloroform (3×30mL). It was then concentrated to a thick oil. The solution was appliedon a Dowex 50 W 8 column (100 g). The fractions containing the productwere concentrated to dryness then recrystallized in a mixture of ethanol(30 mL) and water (2.1 mL). The mixture was cooled slowly at roomtemperature. The solid was collected by suction filtration, rinsed withethanol (2×5 mL) air dried 5 minutes, then 18 hours at 60° C. in thevacuum oven. The desired material was obtained as a fine white solid(3.06 g, 13.7 mmol, 68%). The ¹H and ¹³C NMR and MS were consistent withthe structure.

Preparation of 4-(adamantyl)amino-1-butanesulfonic acid (Compound LI)

1-adamantaneamine hydrochloride (2.67 g, 13.3 mmol) was treated with 1NNaOH (20 mL) and CH₂Cl₂ (3×20 mL). The biphasic solution was shaken. Theorganic extracts were combined, dried with Na₂SO₄, filtered, evaporatedunder reduced pressure and dried in vacuo.

To a solution of 2-adamantanamine (1.87 g, 12.4 mmol) in tetrahydrofuran(THF, 15 mL) was added 1,4-butane sultone (1.76 g, 13.0 mmol). Thesolution was stirred at reflux overnight. The reaction was cooled toroom temperature. The solid was collected by filtration, washed with THF(1×15 mL) and dried in vacuo. A suspension of the solid in EtOH (25 mL)was stirred at reflux for 1 hour. The warm mixture was filtered. Thesolid was dried in vacuo. ¹H NMR (D₂O, 500 MHz) δ ppm 2.92 (m, 2H), 2.82(m, 1H), 2.05 (s, 3H), 1.75 (s, 6H), 1.63 (m, 6H), 1.52 (m, 3H). ¹³C(D₂O, 125 MHz) δ ppm 57.62, 50.30, 39.03, 38.14, 35.09, 28.98, 25.25,21.63. ES-MS 288 (M+1).

Preparation of 4-(octylamino)-1-butanesulfonic acid (Compound LJ)

To a solution of octylamine (2.20 g, 17.0 mmol) in tetrahydrofuran (11mL) was added 1,4-butane sultone (2.30 g, 16.2 mmol). The solution washeated to reflux for 5 hours. The reaction was cooled to roomtemperature. The product formed a gel. A few drops of EtOH were added todissolve the product. The solution was poured in a large excess ofacetone (25 mL). After 5 minutes, a white solid precipitated. The solidwas collected by filtration and dried in vacuo. The product wasdissolved in EtOH and Dowex 50×8 resin (pre-washed, 6 g) was added tothe solution. The suspension was stirred for 15 minutes and the resinwas filtered. The filtrate was evaporated under educed pressure and theproduct was dried in vacuo. Yield: 31%. ¹H NMR (DMSO, 500 MHz) δ ppm8.24 (s (broad), 1H), 2.85 (m, 4H), 2.45 (m, 2H), 1.64 (m, 4H), 1.61 (m,2H), 1.25 (m, 10H), (m, 2H), 0.85 (t, 3H, J=6.8 Hz). ¹³C (DMSO, 125 MHz)δ ppm 51.22, 47.54, 47.38, 31.82, 29.14, 26.59, 26.13, 25.51, 22.95,22.75, 14.64. ES-MS 266 (M+1).

Preparation of 4-(cyclohexylamino)-2-butanesulfonic acid (Compound KM)

To a solution of cyclohexylamine (1.50 g, 15.1 mmol) in tetrahydrofuran(15 mL) was added 2,4-butane sultone (2.04 g, 14.4 mmol). The solutionstirred at reflux for 2 hours. The reaction was cooled to roomtemperature. The solid was collected by filtration and dried in vacuo.Yield: 59%. ¹H NMR (DMSO, 500 MHz) δ ppm 8.50 (s (broad), 1H), 3.02 (m,2H), 2.93 (m, 1H), 2.60 (m, 1H), 1.93 (m, 3H), 1.75 (m, 3H), 1.57 (m,1H), 1.21 (m, 4H), 1.11 (m, 4H). ¹³C (DMSO, 125 MHz) δ ppm 56.23, 53.20,43.09, 29.54, 29.41, 29.39, 25.40, 24.42, 17.23. ES-MS 234 (M−1).

Preparation of 4-[(dl)-1-hydroxy-2-pentyl]amino-1-butanesulfonic acid(Compound LL)

To a solution of DL-2-aminopentanol (1.0 g, 9.7 mmol) in tetrahydrofuran(6 mL) was added 1,4-butane sultone (1.31 g, 9.2 mmol) at roomtemperature. The solution was stirred at reflux for 5 hours. Thereaction was cooled to room temperature. The supernatant was removed andthe solid was dried in vacuo. The white solid was filtered, washed withacetone (2×25 mL) and dried in vacuo. Yield: 45%. ¹H NMR (DMSO, 500 MHz)δ ppm 8.20 (s (broad), 1H), 5.23 (m, 1H), 3.66 (m, 1H), 3.49 (m, 1H),3.02 (m, 1H), 2.91 (m, 2H), 2.46 (t, 2H, J=7.3 Hz), 1.65, (m, 4H), 1.54(m, 2H), 1.38 (m, 2H), 0.88 (t, 3H, J=7.3 Hz). ¹³C (DMSO, 125 MHz) δ ppm58.83, 58.54, 51.23, 44.77, 29.91, 25.56, 23.06, 18.95, 14.48. ES-MS 238(M−1).

Preparation of 3-[(3,4-dimethoxybenzyl)amino]-1-butanesulfonic acid(Compound LM)

To a solution of veratrylamine (1.50 g, 9.0 mmol) in 1,4-dioxane (8 mL)was added 1,4-butane sultone (1.21 g, 8.5 mmol) at room temperature. Themixture was then heated at reflux for 2 hours. The reaction was cooledto room temperature. The solid was collected by filtration, washed withacetone (2×25 mL) and dried on pump. Yield: 18%. ¹H NMR (D₂O, 500 MHz) δ6.96 (m, 3H), 4.04 (s, 2H), 3.74 (m, 6H), 2.95 (t, 2H, J=6.8 Hz), 2.80(t, 2H, J=7.3 Hz), 1.68, (m, 4H). ¹³C (D₂O, 125 MHz) δ ppm 149.19,148.50, 123.82, 123.36, 113.25, 112.17, 55.91, 50.88, 50.24, 46.41,24.55, 21.50. ES-MS 302 (M−1).

Preparation of 4-(adamantan-1-ylamino)-2-hydroxy-1-propanesulfonic acid(Compound KB)

1-adamantaneamine hydrochloride (2.67 g, 14.2 mmol) was treated with 1NNaOH (20 mL) and CH₂Cl₂ (3×20 mL). The organic extracts were combined,dried with Na₂SO₄, filtered, evaporated under reduced pressure and driedin vacuo.

To an 80° C. solution of 1-adamantanamine (2.15 g, 14.2 mmol) in1,4-dioxane (10 mL) and water (5 mL) was added via syringe pump (1 haddition) a solution of 3-chloro-2-hydroxy-propanesulfonic acid, sodiumsalt (1.93 g, 9.7 mmol) in 1,4-dioxane (0.5 mL) and water (10 mL). Thesolution was stirred at reflux overnight. The reaction was cooled toroom temperature. The solvent was evaporated under reduced pressure. Thesolid was suspended in 25% acetone/EtOH. The mixture was heated toreflux for 1 minute. The solid was collected by filtration. The pureproduct crystallized in the filtrate. The product was filtered, washedwith EtOH (2×10 mL), dissolved in water and lyophilized. Yield: 15%. ¹HNMR (D₂O, 500 MHz) δ ppm 4.18 (m, 1H), 3.22 (m, 1H), 3.01 (m, 2H), 2.94(m, 1H), 2.06 (s, 3H), 1.77 (m, 7H), 1.61 (d, 3H), 1.53 (m, 3H). ¹³C(D₂O, 125 MHz) δ ppm 64.23, 57.99, 55.05, 44.10, 38.10, 35.07, 29.03.ES-MS 288 (M−Na (23)).

Preparation of 4-(2-adamantyl)amino-1-butanesulfonic acid (Compound LN)

2-adamantanamine hydrochloride (2.50 g, 13.3 mmol) was treated with 1NNaOH (20 mL) and CH₂Cl₂ (3×20 mL). The organic extracts were combined,dried with Na₂SO₄, filtered, evaporated under reduced pressure and driedin vacuo.

To a solution of 2-adamantanamine (1.06 g, 7.0 mmol) in 1,4-dioxane (6mL) was added 1,4-butane sultone (955 mg, 6.7 mmol). The solution wasstirred at reflux for 5 hours. The reaction was cooled to roomtemperature. The solid was collected by filtration. It was suspended inEtOH (25 mL) and the mixture was heated to reflux for 1 minute beforethe solid was filtered. It was washed with EtOH (1×15 mL) and dried invacuo. Yield: 55%. ¹H NMR (D₂O, 500 MHz) δ ppm 3.29 (m, 1H), 2.97 (m,2H), 2.83 (m, 2H), 2.02 (m, 2H), 1.83 (m, 2H), 1.68 (m, 14H). ¹³C NMR(D2O, 125 MHz) δ ppm 63.07, 50.25, 45.03, 36.55, 36.31, 29.85, 29.05,26.68, 26.41, 24.47, 21.61. ES-MS 286 (M−1).

Preparation of 3-(2-adamantylamino)-2-hydroxy-1-propanesulfonic acid(Compound KJ)

To an 80° C. solution of 2-adamantanamine hydrochloride (2.50 g, 13.3mmol) and sodium hydroxide (586 mg, 14.6 mmol) in 1,4-dioxane (7 mL) andwater (7 mL) was added via syringe pump (1 hour addition) a solution of3-chloro-2-hydroxy-propane sulfonic acid, sodium salt (1.76 g, 8.9 mmol)in 1,4-dioxane (1 mL) and water (9 mL). The solution was stirred at 80°C. for an additional 4 hours. The reaction was cooled to roomtemperature. The solvent was evaporated under reduced pressure. Thesolid was suspended in EtOH (25 mL). The mixture was heated to refluxfor 1 minute. The solid was removed by filtration. The pure productcrystallized in the filtrate. The product was filtered, washed with EtOH(1×10 mL) and dried in vacuo. Yield: 30%. ¹H NMR (D₂O, 500 MHz) δ ppm4.30 (m, 1H), 3.35 (m, 2H), 3.03 (m, 3H), 2.07 (m, 2H), 1.84 (m, 2H),1.75 (m, 4H), 1.65 (d, 6H). ¹³C (D₂O, 125 MHz) δ ppm 63.31, 55.03,49.51, 36.51, 36.33, 36.26, 29.78, 29.23, 28.81, 26.63, 26.38. ES-MS 289(M+1).

Preparation of3-(bicyclo[2.2.1]hept-2-ylamino)-2-hydroxy-1-propanesulfonic acid(Compound KI)

To an 80° C. solution of exo-2-aminonorbornane (910 mg, 8.2 mmol) andsodium hydroxide (242 mg, 6.1 mmol) in 1,4-dioxane (4 mL) and water (4mL) was added via syringe pump (1 hour addition) a solution of3-chloro-2-hydroxy-propane sulfonic acid, sodium salt (1.09 g, 5.5 mmol)in 1,4-dioxane (0.5 mL) and water (5.5 mL). The solution was stirred at80° C. for an additional 5 hours. The reaction was cooled to roomtemperature. The solvent was evaporated under reduced pressure. Thesolid was suspended in EtOH (25 mL). The mixture was heated to refluxfor 1 minute. The solid was recovered by filtration and it was passedthrough an ion exchange column (Dowex 50×8, 100 g, solvent: water). Theproduct was recrystallized in EtOH/water (99/1). Yield: 17%. ¹H NMR(D₂O, 500 MHz) δ ppm 4.25 (m, 1H), 3.25 (m, 2H), 3.01 (m, 4H), 2.39 (m,1H), 2.27 (m, 1H), 1.69 (m, 1H), 1.51 (m, 1H), 1.38 (m, 3H), 1.19 (m,1H), 1.07 (m, 2H). ¹³C (D₂O, 125 MHz) δ ppm 63.66, 63.50, 62.21, 61.98,54.94, 50.11, 50.04, 39.26, 39.21, 36.02, 35.97, 35.91, 35.80, 34.70,34.61, 27.11, 27.08, 26.50, 26.45. ES-MS 250 (M−1).

Preparation of 4-[(3-methylbutyl)amino]-2-butanesulfonic acid (CompoundKH)

To a hot solution of isoamylamine (2.0 g, 22.9 mmol) in tetrahydrofuran(THF, 11 mL) was added via syringe pump (2 hour addition) a solution of2,4-butane sultone (3.1 g, 21.8 mmol in THF (total of 5 mL)). Thesolution was stirred at reflux for an additional 2 hours. The reactionwas cooled to room temperature. The solid was recovered by filtrationand it was washed with THF (25 mL) and acetone (25 mL). The solid wasdissolved in water (20 mL) and Dowex 50×8 (10 g) was suspended in thesolution. The mixture was stirred for 15 minutes and the resin wasfiltered. The solvent was evaporated under reduced pressure. Yield: 28%.¹H NMR (H₂O, 500 MHz) δ ppm 3.07 (t, 2H, J=7.8 Hz), 2.92 (t, 2H, J=7.8Hz), 2.87 (m, 1H), 2.06 (m, 1H), 1.77 (m, 1H), 1.51 (m, 1H), 1.42 (m,2H), 1.18 (d, 3H, J=6.8 Hz), 0.78 (d, 3H, J=6.3 Hz). ¹³C(H₂O, 125 MHz) δppm 53.21, 46.32, 45.37, 34.36, 28.16, 25.35, 21.51, 14.79. ES-MS 224(M+1).

Preparation of 2-hydroxy-3-[(3-methylbutyl)amino]-1-propane sulfonicacid (Compound KK)

To a 80° C. solution of isoamylamine (2.0 g, 22.9 mmol) in 1,4-dioxane(9 mL) and water (3 mL) was added via syringe pump (1 h addition) asolution of 3-chloro-2-hydroxy-propane sulfonic acid, sodium salt (3.04g, 15.3 mmol) in 1,4-dioxane (9.5 mL) and water (0.5 mL). The solutionwas stirred overnight at 80° C. The solvent was evaporated. The productwas passed through an ion exchange column (Dowex 50×8, 100 g, solvent:water). It was recrystallized in absolute EtOH and lyophilized. Yield:27%. ¹H NMR (H₂O, 500 MHz) δ ppm 4.24 (m, 1H), 3.22 (m, 1H), 3.02 (m,5H), 1.49 (m, 3H), 0.79 (d, 3H, J=6.3 Hz). ¹³C(H₂O, 125 MHz) δ ppm63.54, 54.89, 51.53, 46.48, 34.12, 25.46, 21.56, 21.46. ES-MS 226 (M+1).

Preparation of 3-[(dl)-1-Hydroxy-2-pentyl]amino-1-propane sulfonic acid(Compound KL)

To a 80° C. solution of DL-2-amino-1-pentanol (1.0 g, 9.7 mmol) in1,4-dioxane (5 mL) and water (3 mL) was added via syringe pump (1 houraddition) a solution of 3-chloro-2-hydroxy-propane sulfonic acid, sodiumsalt (1.84 g, 9.2 mmol) in 1,4-dioxane (6 mL) and water (0.5 mL). Thesolution was stirred overnight at 80° C. The solvent was evaporated. Theproduct was passed through an ion exchange column (Dowex 50×8, 100 g,solvent: water). The product was dissolved. It was recrystallized inabsolute EtOH and lyophilized. Yield: 27%. ¹H NMR (H₂O, 500 MHz) δ ppm4.26 (m, 1H), 3.77 (m, 1H), 3.32 (m, 1H), 3.24 (m, 1H), 3.03 (m, 3H),1.54 (m, 2H), 1.29 (m, 2H), 0.81 (t, 3H, J=7.3 Hz). ¹³C(H₂O, 125 MHz) δppm 63.69, 63.60, 59.49, 59.38, 58.81, 58.36, 54.98, 48.68, 48.27,29.32, 28.85, 18.40, 18.38, 13.12. ES-MS 242 (M+1).

Preparation of 4-(1H-benzimidazol-2-ylthio)-2-butanesulfonic acid(Compound NE)

To a hot solution of 2-mercaptobenzimidazole (2.0 g, 13.3 mmol) in1,4-dioxane (12 mL) and water (3 mL) was added via syringe pump (1 houraddition) a solution of 2,4-butane sultone (1.80 g, 12.7 mmol in1,4-dioxane (total of 3 mL)). The solution was stirred at reflux for anadditional 3 hours. The solid was collected by filtration. It was washedwith acetone (2×20 mL) and dried in vacuo. Yield: 86%. ¹H NMR (DMSO, 500MHz) δ ppm 7.64(m, 2H), 7.44 (m, 2H), 3.63 (t, 2H, J=7.3 Hz), 2.68 (m,1H), 2.10 (m, 1H), 1.90 (m, 1H), 1.15 (d, 3H, J=6.8 Hz). ¹³C (DMSO, 125MHz) δ ppm 152.64, 133.32, 125.66, 113.67, 53, 10, 39.72, 33.48, 30.14,16.85. ES-MS 287 (M+1).

Preparation of 4-(cyclohexylamino)-1-butanesulfonic acid (Compound LK)

To a solution of cyclohexylamine (2.0 g, 20.2 mmol) in 1,4-dioxane (13mL) was added 1,4-butane sultone (2.61 g, 19.2 mmol). The solution washeated to reflux for 2 hours. The reaction was cooled to roomtemperature. The solid was collected by filtration, washed with acetone(2×20 mL) and dried in vacuo. Yield: 52%. ¹H NMR (D₂O, 500 MHz) δ ppm2.95 (m, 3H), 2.81 (m, 2H), 1.92 (m, 2H), 1.67 (m, 6H), 1.52 (m, 1H),1.18 (m, 4H), 1.02 (m, 1H). ¹³C (D2O, 125 MHz) δ ppm 57.32, 50.31,44.01, 29.02, 24.84, 24.68, 24.07, 24.55. ES-MS 236 (M+1).

Preparation of 3-[(1-ethyl-1-methylpropyl)amino]-1-propanesulfonic acid(Compound FP)

The flask was closed with a septum and connected to a 20% NaOH scrubberfor the Ritter Reaction. Potassium cyanide (3.25 g, 50 mmol) was addedto acetic acid (13 mL) and the mixture was stirred for 10 min at roomtemperature. A solution of sulfuric acid (7 mL) in acetic acid (6 mL)was added and the resulting suspension was stirred 10 minutes at roomtemperature. The 3-methyl-3-pentanol (5 g, 48.9 mmol) was addeddrop-wise over a 5 minute period. The mixture was stirred at roomtemperature for 4 hours, at which time some chunks of potassium cyanidewere still visible.

Another portion of potassium cyanide (0.6 g, powdered) was added and themixture was stirred for 18 hours at room temperature. The mixture waspurged with nitrogen for 1 h then poured over ice (approx. 50 g). The pHof the solution was adjusted to 9 with the addition of 20% NaOH (use 50%next time to reduce the volume). The layers were separated and theaqueous layer was extracted with ether (1×20 mL). The combined organiclayers were washed with saturated potassium carbonate (1×5 mL) thendried over magnesium sulfate. The ether was evaporated under reducedpressure to afford a yellow oil (4.11 g, 31.8 mmol, 64%). The oil showedto be a mixture of cis and trans formamide but what otherwise pureenough to be used as such. ¹H NMR (500 MHz, DMSO-d6) δ 0.75-0.80 (m,6H), 1.11-1.12 (m, 3H), 1.40-1.54 (m, 2H), 1.66-1.73 (m, 2H), 7.35-7.45(br s and br d, 1H), [7.88 (s) and 8.08 (d, J=11.7 Hz) for 1H); ¹³C NMR(125 MHz, DMSO-d6) δ 7.7, 7.9, 23.4, 24.2, 30.6, 33.8, 55.6, 160.3,163.3

The 1-ethyl-1-methyl-propylformamide (4.00 g, 31.1 mmol) was added to20% NaOH (40 mL). The mixture was heated to reflux for 4 hours then wasleft overnight at room temperature. Toluene (10 mL) was added and thelayers were separated. The organic layer was dried over sodium sulfatethen filtered. The final volume of the filtrate was about 30 mL. It wasused as such in the next step.

A solution of 1,3-propanesultone (2.5 g, 20 mmol) in 2-butanone (10 mL)was added to a solution of 3-methyl-3-ethyl-3-propylamine in toluene(total volume: 30 mL). The mixture was heated to reflux for 5 hours thenwas cooled to room temperature. The solid was collected bysuction-filtration and rinsed with acetone (2×5 mL). The solid was driedovernight at 45° C. in the vacuum oven. The title compound was obtainedas a fine white solid (3.63 g, 16.3 mmol, 33% overall yield). ¹H NMR(500 MHz, DMSO-d6) δ 0.79 (t, J=7.3 Hz, 6H), 1.15 (s, 3H), 1.53-1.59 (m,4H), 1.97-2.00 (m, 2H), 2.89 (t, J=7.1 Hz, 2H), 3.03 (t, J=7.6 Hz, 2H);¹³C NMR (125 MHz, DMSO-d6) δ 6.8, 20.2, 21.9, 27.7, 39.7, 48.2, 63.3ES-MS 224; (M+H)

Preparation of3-({2-hydroxy-1,1-dimethyl-2-(3-methoxyphenyl)ethyl]amino)-1-propanesulfonicacid (Compound NG)

To a cooled solution of sodium methoxide (0.5 M in MeOH, 25 mL1) wasadded via syringe over a 10 minutes period 2-nitropropane (5.0 g, 56mmol). The reaction mixture was stirred at room temperature for 30minutes and recooled before m-anisaldehyde (6.8 mL, 56 mmol) was added.The reaction mixture was stirred at room temperature overnight. Themixture was neutralized with Amberlite IR-120 (strongly acidic). Theresin was removed by filtration and washed with MeOH (2×20 mL). Thefiltrate was evaporated. The resulting oil was purified by flashchromatography: 98% Hexanes/EtOAc to 90% Hexanes/EtOAc, affording thedesired nitro compound (5.70 g, 45%).

To a solution of the nitro compound (5.70 g, 25.3 mmol)) in MeOH (25 mL)was added 6M HCl (25 mL). After cooling to 5° C., zinc powder (8.2 g,125 mmol) was added. The suspension was stirred at 0-5° C. and at roomtemperature overnight. The mixture was filtered on a celite pad. Thefilter cake was washed with MeOH (2×20 mL). The combined filtrates wereevaporated under reduced pressure. The residue was dissolved in EtOAc(40 mL). The mixture was exctracted with 5% NaOH (1×40 mL). The aqueousphase was exctracted with EtOAc (2×40 mL). The combined organic extractswere dried with Na₂SO₄, filtered, evaporated and dried in vacuo toafford the corresponding amine. The amine (2.15 g, 44%) was used withoutfurther purification.

To a solution of amine (2.15 g, 11.0 mmol) in Pinacolone (6 mL) andtoluene (6 mL) was added 1,3-propane sultone (1.28 g, 10.5 mmol). Thesolution was stirred at reflux overnight. The reaction mixture wascooled to room temperature. The solid material was collected byfiltration, was washed with acetone (2×20 mL). The solid was suspendedin EtOH (30 mL). The suspension was stirred at reflux for 1 hour. Themixture was cooled to room temperature. The white solid was filtered,washed with acetone (2×15 mL) and dried in a vacuum oven at 50° C.,affording the title compound, 2.26 g (66%). ¹H NMR (DMSO, 500 MHz) δ ppm8.45 (s (broad), 1H), 7.26 (t, 1H, J=7.9 Hz), 6.89 (m, 3H), 6.30 (d, 1H,J=3.2 Hz), 4.69 (d, 1H, J=3.8 Hz), 3.74 (s, 3H), 3.10 (m, 2H), 2.62 (t,2H, J=6.7 Hz), 2.00 (m, 2H), 1.07 (m, 6H). ¹³C (DMSO, 125 MHz) δ ppm159.24, 142.09, 129.45, 120.80, 114.30, 113.76, 74.16, 62.48, 55.92,50.10, 41.57, 23.30, 21.18, 19.37. ES-MS 316 (M−1).

Preparation of3-{[1-(4-methylbenzyl)cyclohexyl]amino}-1-propane-1-sulfonic acid(Compound NH)

NaOMe (0.5M, 40 mL) was added to nitrocyclohexane (2.58 g, 20 mmol) andthe solution was stirred for 30 minutes then concentrated to afford awhite solid. To this solid was added 4-methylbenzylpyrridinium (6.6 g,13 mmol) and DMSO (20 mL). The mixture was heated at 100° C. for 15hours then cooled to rt and diluted with HCl (1M) and EtOAc. Afterseparation of the two phases, the organic layer was washed twice withHCl (1M) then concentrated to obtain an oily crude. Methanol was addedto precipitate the pyridinium byproduct which was filtered off, and thefiltrate was concentrated and purified by column using Hex:EtOAc 90:10to obtain the desired nitro (still contaminated with the pyridiniumsalt). 2 g, 66% yield.

To a stirred solution of the nitro (2.0 g, 8.58 mmol) in methanol (20mL) was added a spatula of Raney-Ni in water. The suspension washydrogenated under atmospheric pressure of hydrogen for 15 hours (TLCindicates complete consumption of the starting material) then filteredon celite and concentrated under reduced pressure. The crude waspurified by column using CH₂Cl₂:MeOH 80:10 to afford 1.2 g of thecorresponding amine.

To a stirred solution of the amine (800 mg, 3.93 mmol) in THF (8 mL) wasadded 1,3-propane sultone (480 mg, 3.93 mmol). The reaction mixture wasstirred at reflux for 15 hours then cooled to room temperature. Thesolid was collected by filtration and was washed with THF. The solid wassuspended in EtOH (10 mL) and stirred at reflux for 1 hour. Thesuspension was then cooled to room temperature. The solid was collectedby filtration, washed with ethanol and dried under high vacuum to affordthe title compound, 1.1 g (86%). ¹H NMR (500 MHz, DMSO-d₆) δ 1.18-1.78(m, 10H), 2.00 (m, 2H), 2.29 (s, 3H), 2.65 (m, 2H), 2.92 (s, 2H), 3.12(m, 2H), 7.10-7.16 (m, 2H), 8.39 (bs, 2H). ¹³NMR (125 MHz, DMSO-d₆) δ20.74, 21.35, 22.80, 25.05, 31.60, 41.04, 50.24, 61.63, 129.71, 131.38,132.44, 136.80. ES-MS 324 (M−1).

Preparation of3-{[2-(4-methoxyphenyl)-1,1-dimethylethyl]amino}-1-propanesulfonic acid(Compound NI)

To a stirred solution of the phenol (233 mg, 1 mmol) in DMF/THF (2.5mL/2.5 mL) was added MeI (93 uL, 1.5 mmol) followed by K₂CO₃ (276 mg, 2mmol). The suspension was heated at reflux for 15 hours then dilutedwith HCl (1M) and with EtOAc. The organic layer was washed with HCl(1M)then concentrated under high vacuum. The crude was purified by columnusing Hex:EtOAc 90:10 to obtain 215 mg of the methoxy (87% yield).

To a stirred solution of the nitro (300 mg, 1.2 mmol) in methanol (5 mL)was added a small spatula of Raney-Ni in water. The suspension washydrogenated under atmospheric pressure of hydrogen for 3 hours (TLCindicates complete consumption of the starting material) then filteredon prewashed celite and concentrated under reduced pressure. The crudeamine was used as such in the next step.

To the crude amine (240 mg, 1.34 mmol) in solution in THF (3 mL) wasadded 1,3-sultone (181 mg, 1.48 mmol) and the mixture was heated atreflux of THF for 12 hours. The suspension of was cooled down andfiltered. The solid was dried to afford 270 mg of the homotaurin as awhite solid (67% yield). ¹H NMR (500 MHz, D₂O) δ 1.11 (s, 6H), 2.00 (m,2H), 2.67 (m, 2H), 2.80 (m, 2H), 3.12 (m, 2H), 3.74 (s, 3H), 6.90 (m,2H), 7.14 (m, 2H), 8.61 (bs, 2H). ES-MS 272 (M−1). ES-MS 300 (M−1).

Preparation of3-{[2-hydroxy-1,1-dimethyl-2-(4-methylphenyl)ethyl]amino}-1-propanesulfonicacid (Compound NJ)

To a solution of 2-nitropropane (3.0 g, 34 mmol), p-tolualdehyde (4.0mL, 34 mmol) and Tetrahydrofuran (30 mL) was added Amberlyst A-21 (7 g).The reaction mixture was stirred at room temperature for 40 hours. Theresin was removed by filtration and washed with THF (2×20 mL). Thefiltrate was evaporated. The resulting oil was purified by flashchromatography: 98% Hexanes/EtOAc to 90% Hexanes/EtOAc, affording thedesired nitro compound (820 mg, 12%).

A suspension of Pd/C and the nitro compound (820 mg, 3.9 mmol) in EtOAc(10 mL) was stirred under H₂ (1 atm) overnight. The mixture was filteredon a celite pad. The celite was washed with EtOAc (2×15 mL). Thecombined filtrates were evaporated under reduced pressure to afford thecorresponding amine. The amine (470 mg, 67%) was used without furtherpurification.

To a solution of amine (470 mg, 2.6 mmol) in pinacolone (5 mL) andToluene (5 mL) was added 1,3-propane sultone (310 mg, 2.5 mmol). Thesolution was stirred at reflux for 4 hours. The reaction mixture wascooled to room temperature. The solid was collected by filtration, waswashed with acetone (2×10 mL) and dried in vacuo, affording the titlecompound, 196 mg (26%). ¹H NMR (DMSO, 500 MHz) δ ppm 8.46 (s (broad),1H), 7.24 (d, 2H, J=7.8 Hz), 7.16 (d, 2H, J=8.3 Hz), 6.23 (d, 1H, J=3.9Hz), 4.68 (d, 1H, J=3.9 Hz), 3.11 (m, 2H), 2.63 (t, 2H, J=6.8 Hz), 2.29(s, 3H), 2.00 (m, 2H), 1.04 (s, 6H). ¹³C (DMSO, 125 MHz) δ ppm 137.69,137.56, 129.03, 128.45, 74.07, 62.38, 49.91, 41.35, 22.99, 21.39, 20.81,18.76. ES-MS 300 (M−1).

Preparation of3-{[1,1-dimethyl-2-(4-methylphenyl)ethyl]amino}-1-propanesulfonic acid(Compound NK)

NaOMe (0.5M, 20 mL) was added to 2-nitropropane (890 mg, 10 mmol) andthe solution was stirred for 30 minutes then concentrated to afford awhite solid. To this solid was added 4-methylbenzylpyrridinium (3.3 g,15 mmol) and DMSO (15 mL). The mixture was heated at 100° C. for 15hours then cooled to room temperature and diluted with HCl (1M) andEtOAc. After separation of the two phases, the organic layer was washedtwice with HCl (1M) then concentrated to obtain an oily crude product.Methanol was added to precipitate the pyridinium byproduct which wasfiltered off, and the filtrate was concentrated and purified by columnusing Hex:EtOAc 90:10 to obtain the desired nitro but still contaminatedwith the pyridinium salt. 1.32 g, 66% yield.

To a stirred solution of the nitro (700 mg, 3.62 mmol) in methanol (10mL) was added a small spatula of Raney-Ni in water. The suspension washydrogenated under atmospheric pressure of hydrogen for 15 hours (TLCindicates complete consumption of the starting material) then filteredon celite and concentrated under reduced pressure. The crude amine wasused as such in the next step.

To a stirred solution of the amine (550 mg, 3.39 mmol) in THF (8 mL) wasadded 1,3-propane sultone (414 mg, 3.39 mmol). The reaction mixture wasstirred at reflux for 6 hours then cooled to room temperature. The solidwas collected by filtration and was washed with THF. The solid wassuspended in EtOH (5 mL) and stirred at reflux for 1 hour. Thesuspension was then cooled to room temperature. The solid was collectedby filtration, washed with ethanol and dried under high vacuum to affordthe title compound, 210 mg (22%). ¹H NMR (500 MHz, DMSO-d₆) δ 1.13 (s,6H), 2.00 (m, 2H), 2.66 (dd, J=7.0 & 7.0 Hz, 2H), 2.75 (s, 2H), 3.10(dd, J=7.0 & 7.0 Hz, 2H), 6.72 (d, J=8.3 Hz, 2H), 7.00 (d, J=8.3 Hz,2H), 8.60 (bs, 2H), 9.36 (s, 1H). ¹³NMR (125 MHz, DMSO-d₆) δ 23.1, 41.2,43.2, 49.8, 59.4, 115.7, 125.8, 132.3, 157.1. ES-MS 284 (M−1).

Preparation of3-[2-(4-fluorophenyl)-2-hydroxy-1,1-dimethylethyl)amino]-1-propanesulfonicacid (Compound NL)

14 g of washed Amberlyst A21 ion exchange resin were placed in a roundbottom flask to which was added nitropropane (14 mL, 120 mmol) and4F-fluorobenzaldehyde (7.45 g, 60 mmol). The reaction mixture wasstirred overnight then diluted with Et₂O and filtered. The filtrate wasconcentrated under rotavap vaccuo then pump vaccuo by heating at 120° C.to remove the excess of aldehyde. The crude was purified by column usingHex:EA 90:10 to afford 3.3 g (25%) of the Henry-aldol product as acolorless solid.

To a solution of the nitro compound (5 g, 23.5 mmol) in MeOH (100 mL)was added 6M HCl (25 mL). After cooling to 5° C., zinc powder (7.6 g,117 mmol) was added. The suspension was stirred at room temperature for3 hours then filtered on a celite pad. The filter cake was washed withMeOH (2×20 mL). The combined filtrates were evaporated under reducedpressure. The residue was dissolved in EtOAc (40 mL), then K₂CO₃ (1M)was added until basic pH. The organic phase was dried with Na₂SO₄,filtered, evaporated and dried in vacuo to afford 3.5 g (83% yield) ofthe corresponding amine. The amine was used without furtherpurification.

To a stirred solution of the amine (3.3 g, 18.0 mmol) in THF (20 mL) wasadded 1,3-propane sultone (2.2 g, 18.0 mmol). The reaction mixture wasstirred at reflux for 15 hours then cooled to room temperature. Thesolid was collected by filtration, washed with ethanol and with Et₂Othen dried under high vacuum to afford the title compound, 4.25 g (77%yield). ¹H NMR (500 MHz, DMSO-d₆) δ 1.13 (s, 6H), 2.00 (m, 2H), 2.66(dd, J=7.0 & 7.0 Hz, 2H), 2.75 (s, 2H), 3.10 (dd, J=7.0 & 7.0 Hz, 2H),6.72 (d, J=8.3 Hz, 2H), 7.00 (d, J=8.3 Hz, 2H), 8.60 (bs, 2H), 9.36 (s,1H). ¹³NMR (125 MHz, DMSO-d₆) δ 23.1, 41.2, 43.2, 49.8, 59.4, 115.7,125.8, 132.3, 157.1. ¹⁹F (282 MHz, DMSO-d₆) δ −115.15 (m, 1F). ES-MS 304(M−1).

The invention claimed is:
 1. A compound of Formula V:

wherein: A is nitrogen or oxygen; R¹¹ is hydrogen, salt-forming cation,ester forming group, or when A is nitrogen, A and R¹¹ taken togetherform the residue of a natural or unnatural amino acid or a salt or esterthereof, wherein A and R¹¹ taken together are not a leucine residue; nis 3 or 4; aa is an amino acid residue selected from a natural aminoacid residue selected from the group consisting of phenylalanine,tyrosine, or an unnatural amino acid residue selected from the groupconsisting of para-aminophenylalanine, para-bromophenylalanine,ortho-chlorophenylalanine, meta-chlorophenylalanine,para-chlorophenylalanine, meta-chlorotyrosine,3,4-dichlorophenylalanine, 3,4-difluorophenylalanine,3,5-diiodotyrosine, ortho-fluorophenylalanine, meta-fluorophenylalanine,para-fluorophenylalanine, meta-fluorotyrosine, homophenylalanine,homotyrosine, para-iodophenylalanine, 3-iodotyrosine,meta-methyltyrosine, para-nitrophenylalanine, 3-nitrotyrosine,ortho-phosphotyrosine, pentafluorophenylalanine, and phenylglycinewherein said amino acid residue is in the L configuration; m is 1, 2, or3; R¹⁴ is hydrogen or protecting group; R¹⁵ is hydrogen, alkyl or aryl;and pharmaceutically acceptable salts, esters, or prodrugs thereof. 2.The compound of claim 1, wherein n is
 4. 3. The compound of claim 1,wherein n is
 3. 4. The compound of claim 1, wherein m is 1 or
 2. 5. Thecompound of claim 4, wherein m is
 1. 6. The compound of claim 4, whereinm is
 2. 7. The compound of claim 1, wherein A-R¹¹ is a residue of anatural amino acid, or a salt or ester thereof.
 8. The compound of claim7, wherein A-R¹¹ is a phenylalanine residue.
 9. The compound of claim 1,wherein A is an oxygen and R¹¹ is a hydrogen or a salt-forming cation.10. The compound of claim 1, wherein aa is a residue of a natural aminoacid.
 11. The compound of claim 1, wherein (aa)_(m) is a residue ofphenylalanine or phe-phe.
 12. The compound of claim 1, wherein aa is aresidue of an unnatural amino acid.
 13. The compound of claim 1, whereinR¹⁵ is hydrogen or substituted alkyl.
 14. The compound of claim 13,wherein R¹⁵ is arylalkyl.
 15. The compound of claim 13, wherein R¹⁵ ishydrogen.
 16. The compound of claim 1, wherein R¹⁴ and R¹⁵ are bothhydrogen.
 17. The compound of claim 1, wherein said compound is selectedfrom the group consisting of:

and pharmaceutically acceptable salts, esters, or prodrugs thereof.